Gut, 1961, 2, 297

Serum haptoglobin in liver diseaseROGER WILLIAMS, BARBARA E. SPEYER, AND

BARBARA H. BILLING

From the Department of Medicine, Royal Free Hospital, London

SYNOPSIS Serum haptoglobin levels have been measured in 115 cases of widely differing liverdisease. Although low levels were found in some cases of cirrhosis and a number of patients withobstructive jaundice had increased levels, 70% of the values fell within the normal range. Theestimation was of no help in distinguishing between intra- and extrahepatic obstructive jaundice.A characteristic pattern was observed in infective hepatitis, and a falling serum haptoglobin inthe presence of increasing jaundice is diagnostic of the latter condition. The cause of these changesis uncertain. Low levels could not be accounted for by increased red cell breakdown and there wasno correlation with the serum albumin level.

Prednisolone therapy produced a rise in serum haptoglobin level in cirrhosis, which was accom-

panied by an improvement in liver function. Temporary rises in level were observed in intrahepaticobstructive jaundice and were probably due to a non-specific effect of prednisolone. In the lattercondition norethandrolone therapy was often associated with high levels.

Haptoglobin is an a2 globulin which has the specificproperty of combining stoichiometrically with hae-moglobin (Jayle, Boussier, and Tonnelat, 1956).The complex formed cannot pass through theglomerulus and the level of haptoglobin is animportant factor in determining the renal thresholdfor haemoglobin. It is not surprising to find areduced serum haptoglobin level in cases of intra-vascular haemolysis although the mechanism for thereduction in cases of haemolytic disease with extra-vascular erythrocyte destruction is not known (Brusand Lewis, 1959; Owen, de Gruchy, and Smith,1960). The variations in level encountered in otherdiseases are also difficult to interpret, particularlysince fever (Nyman, 1959) and non-specific stresses(Owen, Podanyi, and Smith, 1961a) may causeraised values. Low serum haptoglobin values havebeen reported in some cases of cirrhosis and raisedlevels in obstructive jaundice (Jayle and Vallin,1952; Nyman, 1959; Owen, Mackay, and Got,1959). Recently, Owen et al. (1961b) have stressedthe value of this estimation in the differentialdiagnosis of jaundice; as a single test it provedslightly more effective than the serum alkalinephosphatase, albumin, or flocculation tests.

This paper describes findings in widely differingcases of liver disease. Serial estimations have beenperformed since little attention has previously beenpaid to spontaneous fluctuations, which could

seriously affect the value of a single estimation.The changes in serum haptoglobin observed havebeen related to the degree of haemolysis present andthe effect of prednisolone and other therapy hasbeen assessed.

PATIENTS STUDIED

One hundred and fifteen cases were studied. Thesewere divided into five main groups:-Cirrhosis (40),obstructive jaundice (33), infective hepatitis (27),congenital hyperbilirubinaemia (10), and miscel-laneous (5), as shown in Table I. Two cases ofportal cirrhosis and one alcoholic patient withcirrhosis are included in the group of intrahepaticobstruction since they were deeply jaundiced withall the features of cholestasis. Indeed the twopatients with portal cirrhosis had initially beenmistaken for cases of primary biliary cirrhosis.

METHODS

Haptoglobin was estimated by adding standard methaem-oglobin solution to serum and determining the peroxi-dase activity of the resulting methaemoglobin complexcolorimetrically (Owen, Better, and Hoban, 1960).Haptoglobin concentration is expressed in terms ofbound methaemoglobin (M.Hb). The mean value for31 normal subjects was found to be 81-0 mg.1100 ml.with a range (mean i 2 S.D.) of 8 to 154 mg./100 ml.

2972

Roger Willianis, Barbara E. Speyer, and Barbara H. Billing

TABLE 1PATIENTS STUDIED

Diagnosis

CirrhosisPost-hepatitis or cryptogenicAlcoholicJuvenileHaemochromatosis

Obstructive.jaundice (intrahepatic)Primary biliary cirrhosisChronic chlorpromazine jaundicePortal cirrhosis with cholestasis'Alcoholic cirrhosis with cholestasis'Unexplained

Obstructive jaundite (extrahepatic)CarcinomaStricture

Infective l;epatitis

Congenital hyperbilirubintaeiteicGilbertDubin-Johnson

MiscellaneousSecondary biliary cirrhosisCirrhosis with hepatocellular carcinomaCongenital hepatic fibrosis

Total

'See text.

No. of Cases

196510

1822l2

63

27

82

22 1

115

This agrees reasonably weil with the series of 152 normalsstudied by Smith and Owen (1961), which gave a meanvalue of 93 mg./100 ml. with a range (mean ± S.D.) of13 to 173 mg./100 ml. In some cases the serum was alsoexamined by starch gel electrophoresis using the verticaltechnique of Smithies (1955). The concentrations of theserum protein fractions were determined by the biuretmethod together with electrophoresis and scanning.Serum total bilirubin (Malloy and Evelyn, 1937) andserum alkaline phosphatase (King and Wootton, 1959)have also been measured.Red cell survival and blood volume were determined

by the radioactive chromium technique (Veall and Vetter,1958). The results were expressed in terms of the Crp1 redcell half-life (ti) and were not corrected for chromiumelution. An estimate of peripheral haemoglobin destruc-tion was obtained by multiplying the fraction of red cellsdestroyed daily by the total circulating haemoglobin.Since the chromium red cell half-life is less than the truehalf-life, the calculation overestimates the amount ofhaemoglobin destroyed but should give comparablevalues for different subjects. In a normal person thehaemoglobin destruction rate is approximately 250 mg./kg./day.

RESULTS

1 CIRRHOSIS Serum haptoglobin was reduced (<8mg./100 ml.) in six of the 19 patients with post-hepatitis or cryptogenic cirrhosis. In the others thelevel was usually in the lower part of the normalrange, 11 cases having a value of less than 20 mg./

to

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la

h<

I-u

0sto

POST HEPATITIS& CRYPTOGENIC

ALCOHOLIC

X axe xx

20 60 100 140 IO 220 260 3Cow340S RUM HAPTOGLOBIINrmg.I100sml.s MHb.J

FIG. 1. Serum haptoglobin levels in cirrhosis and obstrt!c-tive jaundicex = Norethandrolone therapy.The hatched area indicates the normal range.

100 ml. (Fig. 1). In general the patients with livercell decompensation, as shown by ascites or jaundice,had a low serum haptoglobin and the well-compensated cirrhotic patients had a higher level.There were, however, many exceptions; for instance,three of the 11 patients with a haptoglobin level ofless than 20 mg./100 ml. were out-patients with awell-compensated cirrhosis and two caszs with levelsof 66 and 71 mg./100 ml. had ascites and impendinghepatic coma. This also applied to the alcoholicpatients with cirrhosis. Two of the six cases studiedhad reduced levels; one of these was clinically well,the other in terminal hepatic coma.The five cases of 'juvenile' cirrhosis (Bearn,

Kunkel, and Slater, 1956) had a reduced serumhaptoglobin. These patients had active disease withraised serum bilirubin and transaminase levels, andfour were subsequently treated with prednisolone asdescribed later.

In idiopathic haemochromatosis the serum hapto-globin level was within the normal range. Althoughthe patients had a definite cirrhosis histologicallynone showed evidence of liver cell failure androutine liver function tests were normal.

Considering all the cases of cirrhosis together,there was no correlation between the serum albuminand the haptoglobin level (r - 0 113 p > 0 1).2 OBSTRUCTIVE JAUNDICE (INTRAHEPATIC) Thepatients in this group had all been jaundiced for

298

Serum haptoglobin in liver dlisease

some months. The level of serum haptoglobin wasvery variable and not related to the underlyingcause of the cholestasis. In 16 of the 24 cases it waswithin the normal range (Fig. 1). Seven had anincreased concentration. In five patients the serumhaptoglobin was over 200 mg./100m1. and twopatients with primary biliary cirrhosis had levelsof 314 and 324 mg./100ml. respectively. Whenexamined by starch gel electrophoresis those serawith high levels showed a normal haptoglobinpattern. The only patient with a low value hadrepeated readings of 0 mg./100 ml. There was noevidence of an inhibitory factor in the serum and theabsence of serum haptoglobin was confirmed bystarch gel electrophoresis. Clinically she had primarybiliary cirrhosis and differed in no way from theother patients with normal or raised levels. Sheprobably has a genetically determined anhapto-globinaemia (Allison, 1959).

Serial readings were obtained in 12 patients withprimary biliary cirrhosis. In four of these the serumhaptoglobin remained constant. In another four thelevel of serum haptoglobin increased or decreasedwith the serum bilirubin level but in the remainingfour the level fluctuated for no obvious reason.

Since five of the seven patients with raised serumhaptoglobin levels in this group were receivingnorethandrolone (Nilevar) therapy for pruritus, theeffect of this drug on strum haptoglobin wasinvestigated. Two patients were studied before andduring its administration. Their haptoglobin levelsrose from 26 to 256 and from 86 to 218 mg./100 ml.and at the same time they became more jaundiced,their serum bilirubin levels rising from 3.0 to 10.6and from 3-4 to 30 mg./100 ml. respectively; noappreciable changes in serum alkaline phosphatasewere recorded.

Extrahepatic The serum haptoglobin was withinthe normal range with the exception of one patient,who had a raised level of 178 mg./100 ml. This wasa young girl with congenital hepatic fibrosis, inwhom the bile duct had been damaged at operation.One patient with a bile duct stricture and a level of138 mg./100 ml. had a previous reading when hewas not jaundiced of 62 mg./100 ml.

Considering all the cases of intra- and extrahepaticobstructive jaundice together (whether on or offNilevar) no correlation was found between thehaptoglobin level and the height of the serum bili-rubin (r = 0.08, p > 01). There was, however, aslight though significant correlation (r = 0.360,p < 0.05) between the serum concentration ofhaptoglobin and alkaline phosphatase.

3 INFECTIVE HEPATITIS Serial readings were

obtained in seven of the 27 cases and showed a

characteristic pattern. Five of these patients hadbeen treated with prednisolone but the same patternwas observed in the other patients who were notgiven steroids. Fig. 2 shows the course of a patientwho had been jaundiced for one week before admis-sion. The initial serum haptoglobin was low but asthe serum bilirubin fell the haptoglobin level rose.Subsequently, the level fell again. The inverserelationship between serum bilirubin and hapto-globin during the early recovery phase of hepatitis

WEEKS

FIG. 2. Changes in serum bilirubin and haptoglobin levelsin a patient with infective hepatitis, who had beenjaundicedfor one week before admission.

SERUM HAPTOGLOB!N[mgq.1 IOOml. as MH b.]

FIG. 3. The inverse relationship between serum hapto-globin and bilirubin levels during the early recovery phaseof infective hepatitis.* * = prednisolone t..erapy.

299

Roger Williams, Barbara E. Speyer, and Barbara H. Billing

00O;l**:

* 00O0

Hdight of Recovery0n#t Jaundice Early Late

O STEROID THERAPY months. This agrees with results from a separategroup of patients, who had had hepatitis one to twoyears previously, and who showed, in general,higher levels distributed around the normal mean.

A Clinically these patients were well though two had aslightly raised serum bilirubin.

0

0

0

1-2 years

FIG. 4. Serum haptoglobin levels in 27 cases of infectivehepatitis at varying stages of the disease.

is also shown in Fig. 3. Two cases were seen muchearlier in the course of the disease shortly afterjaundice had been noticed. The first haptoglobinreadings were 128 mg./100 ml. and 84 mg./100 ml.but as jaundice increased during the first week ofadmission the serum haptoglobin level fell. Sub-sequently, during recovery the level rose and thenfell again as described above.The results in the remainder of the patients, in

whom serial readings were not performed, havebeen related to the stage of the disease and areshown together with the results in the serial cases(one reading for each stage) in Fig. 4. Although thelevels at each stage showed a wide scatter, as mightbe expected from the different degrees of severity ofthe cases, the variation between stages is consistentwith the sequence described above. It can be seenthat the haptoglobin levels during recovery, althoughwithin the normal range, are below the normal mean.In a few patients who have been followed subse-quently, the level rose slowly during the next few

4 CONGENITAL HYPERBILIRUBINAEMIA The serumhaptoglobin levels, together with relevant data onthese patients, are shown in Table II. Although innone of the cases did the level fall below the lowerlimit of the normal range (8 mg./100 ml.), in four ofthe eight patients with Gilbert's disease the serumhaptoglobin was 20 mg./100 ml. or less. In two ofthese the red cell survival was measured and foundto be normal whereas in two other patients withGilbert's disease, who had higher haptoglobin levels,the survival was slightly reduced.

5 MISCELLANEOUS The two patients with con-genital hepatic fibrosis had low serum haptoglobinlevels (4 and 5 mg./100 ml. respectively). Routineliver function tests in these cases were normal apartfrom a slightly raised serum alkaline phosphatase.The one patient with a secondary biliary cirrhosishad a level of 7 mg./100 ml. In this patient biliaryobstruction had been successfully relieved surgicallybut had nevertheless progressed to the stage ofterminal liver failure. The two patients with cirrhosiswith hepatocellular carcinoma were deeply jaundicedand had serum haptoglobin levels of 116 and 204mg./100 ml.

RELATION WITH INCREASED HAEMOLYSIS

In infective hepatitis no relation has been foundbetween the initial haptoglobin level and the increasedrate of haemolysis present, as measured either byCr5l red cell half-life or by the calculated rate ofperipheral haemoglobin destruction (Table Ill). Theserum haptoglobin level fluctuates so much duringthe course of the hepatitis that any such relation-ship would be unlikely, for the rate of disappearance

3LE IISERUM HAPTOGLOBIN CONCENTRATIONS IN 10 CASES OF CONGENITAL HYPERBILIRUBINAEMIA

Age and Sex Type Haptoglobin Serum Total Bilirubin(mg./100 ml.) (mg./100 ml.)

2125143033162825IS13

M

M

M

M

M

M

M

M

M

F

GilbertGilbertGilbertGilbertGilbertGilbertGilbertGilbertDubin JohnsonDubin Johnson

9121220304158813871

1-51-30-82-31-01-03-62-03-22-1

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0

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300

Serum haptoglobin in liver disease

of the radioactive cells during this same period doesnot change (Williams, Pitcher, and Billing 1962).The calculated peripheral destruction per day

depends not only on the rate of cell breakdown, butalso on the total amount of circulating haemo-globin and consequently, if this is reduced, as inCase 5 (Table III), who was anaemic, increasedhaemolysis may not necessarily indicate an increasein the amount of haemoglobin being destroyed. Theresults show that in cirrhosis and extrahepaticobstruction patients with similar peripheral haemo-globin destruction rates have widely differing hapto-globin levels. A low serum haptoglobin may befound in cirrhosis when there is little increasedhaemolysis (Case 1) and in obstructive jaundice ahigh level may occur (Case 9) when there is amarked increase in the rate of haemoglobin des-truction. The poor correlation between haptoglobinlevel and increased red cell breakdown in congenitalhyperbilirubinaemia has already been described.

EFFECT OF PREDNISOLONE THERAPY

There is some evidence that giving corticosteroidproduces a rise in serum haptoglobin (Brus anidLewis, 1959). The main difficulty in assessing theresults in patients with liver disease is therefore todetermine which changes are directly due to a non-specific effect of prednisolone and which aresecondary to any improvement in liver function thatmay result. The latter appeared to be the moreimportant factor in the cases of active juvenilecirrhosis treated with prednisolone (Table IV).Three of these showed a definite rise in serumhaptoglobin level and in each case liver functionalso improved with a fall in serum transaminase andserum bilirubin levels. In the fourth case, however,there was no improvement and here the serumhaptoglobin remained low.

In obstructive jaundice the problem is simpler, foralthough giving prednisolone may produce a fall in

TABLE IIISERUM HAPTOGLOBIN LEVEL RELATED TO RATE OF RED CELL BREAKDOWN

AND PERIPHERAL HAEMOGLOBIN DESTRUCTION.

Case Diagnosis Serum TotalBilirubin(mg./100 ml.)

Haemoglobin Cr5' Red Cell Haemoglobin Serum(g./100 ml.) Half-life Destruction Haptoglobin

(days) (mg./kg./day) (mg./100 ml.)

CirrhosisCirrhosisCirrhosisCirrhosisIntrahepatic obstructive jaundiceIntrahepatic obstructive jaundiceIntrahepatic obstructive jaundiceIntrahepatic obstructive jaundiceExtrahepatic obstructive jaundiceInfective hepatitisInfective hepatitisInfective hepatitisInfective hepatitis

Congenital hyperbilirubinaemiaCongenital hyperbilirubinaemiaCongenital hyperbilirubinaemiaCongenital hyperbilirubinaemia

1-11-66-37.4

20-213-015-340-831-06-813-919-613-1

1-52-31-11-0

11-814-110-110-09-611-912-47.7

12-214-310-111-213-9

13-915-515-415-1

25241813142118131222171423

25252118

259350426453278363407447527270285304375

338270357410

'These are the same case numbers as in Table II.

TABLE IVSERUM HAPTOGLOBIN AND BILIRUBIN LEVELS IN FOUR CASES OF ACTIVE.

JUVENILE' CIRRHOSIS BEFORE AND AFTER PREDNISOLONE THERAPY.

Serum Total Bilirubin (mg./100 ml.)

Before After

Serum Haptoglobin (mg./100 ml.)

Before

Duration of Therapy

After

1-01-0

37

2-0 812-3 3

301

2345678910111213

1141516'

4113

114164184162178129238910

9203041

Case

234

3.08-04.56-3

2349434

4 months4 months

. 3 weeks5 weeks

Roger Willianms, Barbara E. Speyer, and Barbara H. Billing

36 76. 114 155 148 127 .82 0.PREDNISOLONE

FIG. 5. Starch gel electrophoresis of sera from a patientwith intrahepatic obstructive jaundice showing an unalteredhaptoglobin pattern despite marked variation in level withprednisolone therapy.

serum bilirubin, this is not very marked and theeffect is purely temporary. Prednisolone was givento five of the patients with chronic intrahepaticobstruction. In one, the haptoglobin level wasalready falling and continued to do so despite pred-nisolone therapy. Two others showed a slight risein serum haptoglobin coincident with a slight fallin serum bilirubin, these changes being reversedwhen treatment was discontinued. The remainingtwo showed a marked and rapid rise in serum hapto-globin (36 to 155 mg./100 ml. over eight days, and41 to 102 mg./100 ml. over two weeks respectively),whereas the serum bilirubin fell only slightly in thefirst and rose steadily in the second patient through-out the period of observation. Subsequently therewas an equally rapid fall, though steroid therapy wascontinued in one patient. The serum of one of thesewas subjected to starch gel electrophoresis and itwas found that the haptoglobin pattern remainedunaltered in spite of changes in serum concentrationranging from 10 to 155 mg./100 ml. (Fig. 5). Itseems very likely that the response in these twopatients was simply a non-specific effect of predni-solone on haptoglobin level.Three cases of extrahepatic obstructive jaundice

had steroid therapy. Two showed no change while thethird had a slight rise in serum haptoglobin to-gether with a slight fall in serum bilirubin, theresponse being similar to that seen in some of thecases of intrahepatic obstruction described above.The effect of steroid therapy in infective hepatitis

is also difficult to assess. There the serum hapto-globin rises markedly as the serum bilirubin begins

to fall, this occurring with and without prednisolonetherapy. The extent of the rise, however, is probablyinfluenced by prednisolone as suggested by Fig. 3,although this is difficult to assess since the twogroups were not of comparable severity and thehigher levels reached in the treated cases maysimply reflect the greater severity of disease.

DISCUSSION

Seventy per cent of the 73 patients with cirrhosis andobstructive jaundice had values falling within thenormal range so that an isolated haptoglobinestimation is of limited value in the diagnosis ofthese conditions. Nevertheless, certain trends areapparent and the finding of an abnormally low levelin the presence of liver disease is very suggestive ofcirrhosis. This may be of considerable diagnostichelp in the well-compensated patient, who showsfew clinical signs. Nevertheless, there are manyexceptions and the association in some patients offrank, decompensated liver disease with normallevels is unexplained. In obstructive jaundice theestimation seems of less value, for with one exceptionpatients with extrahepatic obstruction had levelswithin the normal range. This is contrary to thefindings of Owen et al. (1961b) who reported that81 % of their patients showed values over 150 mg./100 ml. However, our series is mainly composed ofpatients with carcinomatous obstructions whereasthe majority of Owen's patients had obstruction dueto gall stones. Nyman (1959) also found that it wasonly in the latter type of case, in which cholangitis ispresent, that the level was raised. The serum hapto-globin is of little value in distinguishing betweenintra- and extrahepatic obstructive jaundice, a pointwhich has not been stressed before. It may, however,be of considerable value in the sometimes difficultdifferentiation between infective hepatitis and ob-structive jaundice. A falling haptoglobin level in thepresence of rapidly increasing jaundice is diagnosticof hepatitis (Owen et al., 1961b).The mechanism of these changes in liver disease

is uncertain. Owen and his colleagues (1959) foundsome correlation between the haptoglobin level andthe serum albumin suggesting that the low levels wererelated to impaired hepatic synthesis of protein.This was not found by Nyman (1959) and in thepresent series there was no correlation with serumalbumin. There does, however, appear to be arelation with the activity of the disease. The patientswith active juvenile cirrhosis in whom the hapto-globin level rose with steroid therapy also showedimprovement in liver function. Also, the majority ofthe decompensated cirrhotics had low levels whereasthe patients with alcoholic cirrhosis or haemo-

302

Serunm haptoglobin in liver disease 303

chromatosis, who had a quiescent type of lesion, hada serum haptoglobin within the normal range. Lowlevels, however, were found in two cases ofcongenitalhepatic fibrosis, a condition in which there is littledisturbance of liver function. In any case, there is asyet no direct evidence that the liver is the source ofhaptoglobin and the dramatic rises observed fol-lowing infection and surgery have suggested a pos-sible origin in connective tissue (Jayle and Boussier,1955).

It is clear also that the low serum haptoglobin incirrhosis cannot be attributed to haemolysis. IndeedWilliams et al. (1962) have shown that increasedhaemolysis is common to all forms of jaundice,including that due to biliary obstruction, in whichthe haptoglobin level may be normal or high.Another possible explanation is altered oestrogenmetabolism. Administration of these steroids hasbeen shown to lower the serum haptoglobin (Borglinand Nyman, 1961) and it is known that their con-jugation and excretion in liver disease is sometimesimpaired (Cameron, 1957).There was a slight, though significant, correlation

between the level of serum haptoglobin and alkalinephosphatase. This suggests that the higher level inobstruction might be related to biliary retention.This presumes that haptoglobin is normally excretedin bile but in preliminary experiments we have onlybeen able to demonstrate the presence of hapto-globin in bile in one case and this was a patient withan infection, who had a serum level of over 500mg./100 ml. If biliary retention were a factor onewould also expect a correlation with serum bilirubinwhich was not found. It is germane to consider herethe effect of norethandrolone (Nilevar) therapy, aknown cause of intrahepatic cholestasis. Cases ofprimary biliary cirrhosis receiving norethandrolonedid tend to have higher serum haptoglobin valuesand the administration of this drug to two earlycases caused a definite rise in level. This was ac-companied by a rise in serum bilirubin althoughthe serum alkaline phosphatase was unaltered. Wecannot, therefore, be sure whether the rise inhaptoglobin with norethandrolone is a secondaryresult of increased biliary retention or whether thisdrug has a non-specific effect similar to that ofprednisolone. The abrupt and temporary rises inserum haptoglobin noted in some of the cases ofintrahepatic obstruction are almost certainly due toa non-specific effect of prednisolone.The alterations in serum haptoglobin in infective

hepatitis are of interest. The fall in level as jaundiceincreases and the sudden rise with the onset ofrecovery are difficult to explain. If the rise were dueto improvement in liver function then it is surprisingthat the level subsequently falls. Although the extent

of the rise was greater in the patients on prednisolonetherapy these were the most severe cases, and adefinite increase occurred at this stage even in thepatients who did not receive corticosteroids.Relatively few of these cases had serial estimations;Nyman (1959) has, however, reported a similarpattern in some of her cases of sporadic hepatitis.

In conclusion it is clear that there is as yet nosatisfactory explanation for the changes in serumhaptoglobin observed in differing types of liverdisease. However, it is possible that turnoverstudies using haptoglobin labelled with radioactiveiodine may help to elucidate this problem.

We should like to thank Professor Sheila Sherlock forher encouragement and advice. The work was supportedby the Medical Research Council and Miles LaboratoriesInc.

REFERENCES

Allison, A. C. (1959). Genetic control of human haptoglobin synthesis.Nature (Lond.), 183, 1312-1314.

Bearn, A. G., Kunkel, H. G., and Slater, R. J. (1956). The problemof chronic liver disease in young women. Amer. J. Med., 21,3-15.

Borglin, N. E., and Nyman, M. (1961). The effect of estrogens on thehaptoglobin level in the blood. Scand. J. clin. Lab. Invest., 13,107-115.

Brus, I., and Lewis, S. M. (1959). The haptoglobin content of serumin haemolytic anaemia. Brit. J. Haemat., 5, 348-355.

Cameron. C. B. (1957). The liver and steroid hormone metabolism.Brit. med. Bull., 13, 119-125.

Jayle, M. F., and Boussier, G. (1955). Les s6romucoldes du sang,leurs relations avec les mucoproteines de la substance fonda-mentale du tissu conjonctif. Expos. ann. Biochim. mdd., 17,157-194.

, and Tonnelat, J. (1956). Isolement a partir du s6rumhumain de la combinaison hemoglobine-haptoglobine al'6tat homogene. Bull. Soc. Chim. biol. (Paris), 38, 343-349.and Vallin, J. (1952). Variations de la formule prot6ique duplasma au cours des affections Mpatiques. Sem. Hop. Paris, 28,3133-3138.

King, E. J., and Wootton, I. D. P. (1959). Micro-analysis in MedicalBiochemistry, 3rd ed. (reprint), pp. 82-84. Churchill, London.

Malloy, H. T., and Evelyn, K. A. (1937). The determination of bili-rubin with the photoelectric colorimeter. J. biol. Chem., 119,481-490.

Nyman, M. (1959). Serum haptoglobin: methodological and clinicalstudies. Scand. J. clin. Lab. Invest., 11, Suppl. 39.

Owen, J. A., Better, F. C., and Hoban, J. (1960). A simple methodfor the determination of serum haptoglobins. J. clin. Path.,13, 163-164.Gruchy, G. C. de, and Smith, H. (1960). Serum haptoglobins inhaemolytic states. Ibid., 13, 478-482.Mackay, 1. R., and Got, C. (1959). Serum haptoglobins inhepatobiliary disease. Brit. med. J., 1, 1454-1457.Podanyi, R., and Smith, H. (1961a). The effect of surgery onserum haptoglobins. Proc. Ass. Clin. Biochem., 1, 89-91.

*___,___ - (1961b). Serum haptoglobins and other tests in thediagnosis of hepatobiliary jaundice. Clin. Sci., 21, 189-197.

Smith, H., and Owen, J. A. (1961). The determination of haptoglobinsin normal human serum. Biochem. J., 78, 723-728.

Smithies, 0. (1955). Zone electrophoresis in starch gels: Groupvariations in the serum proteins of normal liuman adults.Ibid., 61, 629-641.

Veall, N., and Vetter, H. (1958). Radioisotope Techniques in ClinicalResearch and Diagnosis. Butterworth, London.

Williams, R., Pitcher, C. S., and Billing, B. H. (1962). Red cellsurvival in infective hepatitis and other forms of jaundice.In preparation.