1007LEADING ARTICLES

Haptoglobins

THE LANCETLONDON 12 MAY 1962

HAPTOGLOBINS are a family of plasma-proteins thathave the distinctive property of combining with haemo-globin. They were discovered in Paris in 1938 by M. F.JAYLE/ who noticed that the peroxidase activity ofhaemoglobin is greatly enhanced by addition of serumand showed that this is due to combination of haemo-

globin in stoichiometric proportions with an a2 globulincomponent to which he gave the name haptoglobin (fromthe Greek &ït’t"É:ou, to hold fast). Estimation of

peroxidase activity either by spectrophotometry 2 or bya photometric method suitable for clinical laboratories 3is still the most satisfactory way of measuring hapto-globin in serum, particularly when it is present only insmall amounts.Later work by JAYLE and his colleagues 4 led to the

isolation from two people of two different haptoglobins,one with a higher molecular weight than the other.Quite independently, SMITHIES 5 in Toronto found that,when sera from human subjects were submitted toelectrophoresis in starch gels, individual differences wereapparent. It later emerged that the proteins showingvariation were haptoglobins, and that on the basis ofhaptoglobin typing human sera could be placed into oneof three main groups-1-1, 2-1, and 2-2. These threetypes are inherited as a result of segregation of a singlepair of genes-Hpl and Hp2.6 s Much evidence hasaccumulated that this is the usual mode of inheritanceof haptoglobins, although occasionally segregation is

atypical. Several other phenotypes have been describedņHpO - in which demonstrable haptoglobins are lack-ing, the Carlsberg type 7, the modified 2-1 type,9 and theJohnson type 9. Furthermore, CONNELL et al.lO haveshown that the Hpl-1 phenotype is, in fact, composed oftwo different types. When purified haptoglobins weredegraded with mercaptoethanol and urea, and submittedto electrophoresis in starch gels containing urea, com-ponents common to all phenotypes as well as com-

ponents with genetic specificity were obtained. The

specific components from type 1-1 were found to have1. Polonovski, M., Jayle, M. F. C.R. Soc. Biol., Paris, 1938, 129, 457.2. Connell, G. E., Smithies, O. Biochem. J. 1959, 72, 115.3. Owen, J. A., Better, F. C., Hoban, J. J. clin. Path. 1960, 13, 163.4. Jayle, M. F., Boussier, G. Exp. ann. biochem. méd. 1955, 17, 157.5. Smithies, O. Biochem. J. 1959, 71, 585.6. Smithies, O., Walker, N. F. Nature, Lond. 1956, 178, 694.7. Galatius-Jensen, F. The Haptoglobins: a Genetical Study. Copenhagen,

1960.8. Allison, A. C., Blumberg, B. S., ap Rees, W. Nature, Lond. 1958, 181,

824.9. Smithies, O., Connell, G. E. in Ciba Foundation Symposium on Bio-

chemistry of Human Genetics (edited by G. E. W. Wolstenholme andC. M. O’Connor); p. 178. London, 1960.

10. Connell, G. E., Dixon, G. H., Smithies, O. Nature, Lond. 1962, 193, 505.

either a -fast (HpIF) or a slow (HpIS) electrophoreticmobility, and the presence of these products was foundto be controlled by allelic genes-HpIF and HpIS. Thisis of general interest since it shows that apparentlyidentical gene products may include concealed differ-ences that can be revealed by appropriate experimentalmanipulation.The haptoglobins exemplify another point of general

interest in biochemical genetics 11: the gene products inheterozygotes (2-1 and modified 2-1 types) are

apparently different from those in either homozygoustype (1-1 or 2-2), which is at first sight difficult to

reconcile. with current ideas on the genetic control ofprotein synthesis. The difficulty was removed whenALLISON,12 and SMITHIES and CONNELL,9 found thatthe heterozygous-type proteins are produced by co-polymerisation of the primary gene products. The factthat one type of haptoglobin (2-2) polymerises whileanother type (1-1), synthesised under the control of anallelic gene, does not, illustrates an important way inwhich gene mutation can change the structure and pro-perties of proteins. Many mutations bring about singleaminoacid substitutions in the protein products of thegenes concerned, as the work of INGRAM et al.I3 on

haemoglobins shows so beautifully. But certain amino-acid substitutions have a special effect: as in the Hp2example, they allow altered molecules to polymerise-aprocess which may affect their properties considerably.Thus, certain enzymes are active only as polymers, andothers show altered substrate specificity with depoly-merisation. TOMKINS et a1.14 have found that the highlypolymerised (tetrameric) form of the enzyme, glutamicdehydrogenase, catalyses the oxidation of glutamic acidwhile the depolymerised (monomeric) form catalysesoxidation of alanine. No difference in enzymic (per-oxidase) activity is apparent between polymerised andunpolymerised haptoglobin molecules, but some suchdifference in other properties may help to explainwhy all human populations so far tested remaindivided or polymorphic for the various haptoglobinphenotypes.I5

Besides being of genetic importance, studies of hapto-globins have contributed results of physiological andclinical interest. The most striking property of hapto-globins is their ability to combine with hxmoglobinliberated into the plasma. Combination also takes placewith isolated globin.I6 The combining power is greatestin type 1-1 (average 136 mg. haemoglobin per 100 ml.plasma), intermediate in type 2-1, and least in type2-2.16 The coincidence between the hxmoglobin-combining power of haptoglobins and the so-called" renal threshold" for haemoglobin, led LAURELL andNYMAN 17 and ALLISON and Ap REES 18 independently toformulate a new explanation for the phenomenon, in11. See Lancet, March 3, 1962, p. 469.12. Allison, A. C. Nature, Lond. 1959, 183, 1313.13. Ingram, V. M. Hemoglobin and its Abnormalities. Springfield, Ill.,

1961.14. Tomkins, G. M., Yielding, K. L., Curran, J. Proc. Nat. Acad. Sci.,

Wash. 1961, 47, 270.15. Barnicot, N. A. in Genetical Variation in Human Populations (edited by

G. A. Harrison); p. 41. Oxford, 1961.16. Nyman, M. Scand. J. clin. Lab. Invest. 1959, 11, suppl. 39.17. Laurell, C. B., Nyman, M. Blood, 1957, 12, 493.18. Allison, A. C., ap Rees, W. Brit. med. J. 1957, ii, 1137.

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place of the previously held concept of a limit to renaltubular reabsorption of hxmoglobin. According to thisinterpretation, haemoglobin bound to haptoglobin doesnot pass into the glomerular filtrate, which free hxmo-globin does. Hence haemoglobinuria will occur onlywhen the haemoglobin concentration in the bloodexceeds the combining capacity of the haptoglobins.This interpretation, which explains puzzling featuresabout the renal threshold for haemoglobin, is now

generally accepted.19 Furthermore, it is only whenthe haemoglobin concentration in the plasma exceeds thecombining capacity of haptoglobins that some ofthe hxm groups are transferred to albumin, forming thecompound methaemalbumin. The only known exceptionto this rule is methaemalbuminaemia in acute pancreatitis,in which the haemoglobin-binding capacity of hapto-globins is not always saturated.2° It is supposed thatpancreatic enzymes liberate from extravasated bloodhsem groups that are absorbed into the blood-stream andcombine directly with albumin.

After release of haemoglobin into the plasma, thehaptoglobin-haemoglobin complex is removed in a fewhours, probably mainly by the liver. 1 7 21 When intra-vascular release of haemoglobin is continuous, as in manyhsemolytic ansemias, the rate of removal of the complexmay exceed the rate of replacement of haptoglobin, sothat the haptoglobin in plasma may fall to low levels orbe altogether undetectable.ls 22 ALLISON 22 reportedthat in sickle-cell anaemia and thalassaemia majorhaptoglobins are usually not detectable, whereas in thesickle-cell trait the level is normal. This has been con-firmed by LATHEM and JENSEN 23 and WHITTEN.24WHITTEN has shown, moreover, that sickle-cell homozy-gotes retain the capacity to synthesise haptoglobin,which may be demonstrable when production of sicklecells is suppressed after repeated transfusion, and alsoduring infections and thrombotic crises. The increasedtubular reabsorption of haemoglobin in sickle-celldisease was not found to impair function of the proximalconvoluted tubules.

Whether haemoglobin binding by haptoglobins hasany physiological significance is not clear, although thesuggestion 18 that haptoglobins thereby prevent ironloss from the body after heemolysis-for instance, duringmalarial attacks-may have relevance in unsophisticatedcommunities with marginal iron intakes. It is equallyobscure why plasma haptoglobin levels should rise con-siderably with even quite trivial tissue damage or

inflammation. This situation has been reproducedexperimentally in rabbits by turpentine injections,25 andthe evidence suggests that haptoglobin can be quicklymobilised from some extravascular reservoir. Althoughwe now know a great deal about the structure and

genetic control of haptoglobins, we still have very littleindication of what they do in the body.19. Lathem, W. J. clin. Invest. 1959, 38, 652.20. Northam, B. E., Rowe, D. S., Winstone, N. E. Brit. med. J. Jan. 27,

1962, p. 260.21. Murray, R. K., Connell, G. E., Pert, J. H. Blood, 1961, 17, 45.22. Allison, A. C. Proc. R. Soc. Med. 1958, 51, 645.23. Lathem, W., Jensen, W. N. Blood, 1960, 14, 1047; J. Lab. clin. Med.

1962, 59, 137.24. Whitten, C. F. New Engl. J. Med. 1962, 266, 529.25. Murray, R. K., Connell, G. E. Nature, Lond. 1960, 186, 86.

Achalasia: Dilatation or Cardiomyotomy?" I prepared for him an instrument like a rod of whalebone

with a little round button of sponge fixed to the top of it; thesick man having taken down meat and drink into his throat,presently putting this down in the oesophagus, he did thrust itdown into the ventricle.... "

So wrote THOMAS WILLIS in 1679 about the treatmentof a patient with achalasia.1ELLIS has described some aspects of the pathology of

achalasia which help to define what different forms oftreatment are likely to achieve. ELLIS and TROUNCE 3

applied cholinergic and adrenergic drugs and their

antagonists to strips of oesophageal muscle removed atoperation from normal and achalasic subjects. Theyfound evidence of cholinergic receptors in the musclewhich were stimulated by acetylcholine, the responsebeing blocked by atropine and potentiated by eserine.These receptors are present in both circular and longi-tudinal muscle from the body of the normal and theachalasic oesophagus; but the postganglionic nerves,which activate these receptors and are stimulated bynicotine, are apparently defective in the body of thecesophagus in achalasia. This defect may account for thefailure of coordinated motor activity and for the hyper-sensitivity to administered cholinergic drugs which

produce violent spasmodic contractions of the mso-

phagus, often associated with considerable pain. The

sphincter segment, on the other hand, has two types ofreceptors. In the longitudinal muscle of both normal andachalasic subjects, they found cholinergic receptorswhich responded to stimulation of the postganglionicfibres with nicotine. In the circular muscle of the

sphincter, however, ELLIS and TROUNCE found adrener-gic receptors, which were stimulated by noradrenalineand had their response modified by phentolamine. Inthe normal oesophagus the postganglionic nerve-fibres,stimulated by nicotine, activated these adrenergicreceptors, producing relaxation of the muscle, but inachalasia this relaxation process was apparently defec-tive.These 4 and other studies suggest that the changes in

motility in achalasia result from: (1) a defect of post-ganglionic fibres in the body of the oesophagus wherebyperistalsis is lost; and (2) a defect of the adrenergicrelaxation process of the sphincter. Treatment, there-fore, must be either pharmacological or mechanical.Cholinergic drugs worsen the symptoms, and adrener-gic drugs are without clinically useful effects Inhalationof octyl nitrite is useful in relaxing the sphincter, but itseffect lasts only a few minutes and it does not restorepropulsive activity to the body of the oesophagus. Of themechanical methods bougienage, introduced by WILLIS,produces relief for an hour or two. Dilatation with airor water filled bags, or STARK’s mechanical dilatory isparticularly favoured by physicians since these intru-ments may be used by the endoscopist, requiring little1. Quoted by F. G. Ellis. Ann. R. Coll. Surg. Engl. 1962, 30, 155.2. Ellis, F. G. ibid.3. Ellis, F. G., Trounce, J. R. Brit. J. Surg. 1960, 47, 466.4. Adams, C. W. M., Brain, R. H. F., Ellis, F. G., Kauntze, R., Trounce,

J. R. Guy’s Hosp. Rep. 1961, 110, 191.5. Sleisenger, M. H., Steinberg, H., Almy, T. P. Gastroenterology, 1953,

25, 333.6. Stark, H. Schweiz. med. Wschr. 1923, 53, 613.