Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Postgrad. med. J. (February 1969) 45, 86-106.
Bilirubin and red cell metabolism in relation to neonatal jaundice
TIMOS VALAES M.D. (Bristol) D.C.H. The 'Queen Anna-Maria' Institute of Child Health, Athens, Greece
Introduction newborn period this amount is in full-term infants For over half a century neonataljaundice has been 21-25%, and in prematures 30% of the total the subject of clinical and laboratory investigations. bilirubin load (Vest, 1967). In assessing the daily Spectacular progress has been made during these load of bilirubin in the newborn period two more years. Work on neonatal jaundice has contributed to factors should be considered: (a) The infant begins our understanding of physiological and pathological its life with an increased concentration and total mechanisms in the areas of foeto-maternal relation- mass of haemoglobin per kg of body weight; and ships, of bile pigment biochemistry and metabolism, (b) the life-span of the red cells of the newborn etc. Notwithstanding these fundamental contribu- (term, and particularly of the pre-term one) is tions the exact mechanism of even the most common shortened (80 days approximately) compared with type of neonatal jaundice, the so-called 'physio- that of the adult (120 days) (Vest & Grieder, 1961; logical jaundice', is still beyond our grasp. More- Kaplan & Hsu, 1961; Garby, Sjolin & Viulle, 1964; over, in recent years disturbances of red-cell meta- Pearson, 1967; Maisels, Pathak & Nelson, 1968). bolism have been recognized as important causes of severe neonatal jaundice in some ethnic groups, Bilirubin excretion by copyright. while racial and geographical differences in the Three steps have been recognized in bilirubin degree of non-specific neonatal hyperbilirubinaemia excretion: (a) uptake of bilirubin by the liver cells; have added to the complexity of the subject. These (b) conjugation of bilirubin with glucuronic acid to latter aspects of neonatal jaundice are the main form a diglucuronide ester of bilirubin; and (c) subject of the present review. excretion of conjugated bilirubin by the liver cells To identify some areas relevant to our main dis- into the lumen of biliary canaliculi. cussion a brief outline of bilirubin metabolism in The first step has not been studied in newborn the newborn follows. For more details the reader is animals. In mature animals it is not the referred to excellent recent reviews & rate-limiting many (Billing step (Grodsky, 1967). Conjugation of bilirubin takes http://pmj.bmj.com/ Lathe, 1958; Lathe, Claireaux & Norman, 1958; place mainly in the liver. Uridine diphospho- Lucey, 1960; Zuelzer & Brown, 1961; Brown, 1962; glucuronic acid is the donor substance and the Lester & Schmid, 1964; Cracco, Dower & Harris, enzyme glucuronyl transferase catalyses the transfer 1965). of glucuronic acid to bilirubin (Grodsky & Carbone, 1957; Lathe & Walker, 1957; Schmid, Hammaker Metabolism of bilirubin in the newborn & Axelrod, 1957). It is now accepted that the new- Bilirubin production born period is characterized by a relative insuffic- Jaundice always means that the production of iency of the enzymatic mechanism of bilirubin on September 27, 2021 by guest. Protected bilirubin exceeds excretion with the result that the conjugation (Lathe & Walker, 1957; Brown & pigment accumulates in the plasma and the tissues. Burnett, 1957; Brown, Zuelzer & Burnett, 1958; Bilirubin production basically means red-cell destruc- Dutton, 1958, 1959; Vest, 1958). This functional im- tion-haemolysis-either at the end of their normal maturity of the liver of the newborn is more pro- life or at an earlier stage through a variety of mecha- nounced in infants with shortened gestation. The nisms which shorten red-cell life-span. A small degree of this insufficiency is not amenable to direct proportion of the daily production of bilirubin is not measurement but there is indirect evidence of a derived from the degradation of haemoglobin liber- considerable individual variation. This can be ated from mature red cells but from other sources exemplified by the number ofinfants with haemolytic (Gray, Newberger & Sneath, 1950; London et al., disease of the newborn (HDN) from Rhesus incom- 1950). In the adult the amount of bilirubin derived patibility who develop severe anaemia with little from sources other than the break-down of red cells or no jaundice and by the variable degree of hyper- is approximately 10-15% of the total, while in the bilirubinaemia in infants with similar extravasation Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonataljaundice 87 of blood in cases of giant subaponeurotic cephal- noted that the time-interval was not connected with haematomas (Valaes & Doxiadis, 1968). It has been the maturity of the infant and seemed to be condi- estimated that it takes approximately 3-4 weeks for tioned only by the onset of extrauterine existence. the mature infant to reach adult levels of bilirubin- conjugating capacity (Vest, 1958). The availability Significance ofdefectivefoetal bilirubin conjugation of the donor substance uridine diphosphate glu- Teleologically speaking we can say that the in- curonic acid depends on a complex enzymatic sufficiency of conjugation is an advantage to the process which includes carbohydrate metabolism foetus as it leads to the removal of the bilirubin and a specific enzyme of the soluble fraction of the produced during foetal life by the maternal liver. liver homogenate, UDPGA dehydrogenase, which Experimental work has shown that unconjugated- again is diminished in the neonatal period (Brown lipid-soluble-bilirubin easily crosses the placental et al., 1958; Dutton, 1958, 1959). membrane and enters the maternal circulation even The enzymatic insufficiency, which leads to de- at a very low gradient (Lester, Behrman & Lucey, creased capacity for bilirubin excretion and to the 1963; Wynn, 1963; Grodsky etal., 1963; Schenker, accumulation of indirect, lipid-soluble, bilirubin, is Bushare & Smith, 1967) while conjugated bilirubin the common denominator of all types of neonatal enters the maternal side only in very small amounts jaundice whatever are the other co-existing mecha- (Schenker et al., 1967). Even before this experimental nisms. This transient metabolic immaturity of the evidence, disposal of foetal bilirubin through the newborn explains many of the clinical peculiarities maternal liver was postulated in order to explain the of neonatal jaundice. relatively low concentration of serum bilirubin in The final phase in bilirubin excretion, i.e. the the cord blood of infants with HDN. excretion of conjugated bilirubin by the liver cells An alternative and more plausible explanation for into the biliary canaliculi, is not yet fully understood the low bilirubin-conjugating capacity of the new- (Lester & Schmid, 1964). Work by Arias, Johnson born would be that the placental circulation by & Wolfson (1961) has suggested that this step is a removing the bilirubin produced in foetal life rate-limiting one in bilirubin excretion in the adult removes the stimulus for the development of the by copyright. rat. In foetal guinea-pigs Schenker and his associ- conjugating mechanism which then should be con- ates (Schenker, Dawber & Schmid, 1964) found sidered one of the adaptive enzymatic systems impaired excretion ofconjugated bilirubin suggesting (Sereni & Principi, 1965). That this might be true is that this step is also immature in the newborn period. suggested by a close quantitative study of the There is indirect evidence that this applies also to bilirubin metabolism in cases of severe HDN from human newborns. The most plausible explanation for Rhesus isoimmunization. The fairly good correla- the appearance ofhigh conjugated bilirubin values in tion between cord-blood serum bilirubin values and the course of haemolysis in the newborn period-the the severity of the haemolytic process suggest that in so called 'inspissated bile syndrome-is a dis- spite of the readiness with which bilirubin crosses crepancy between conjugating and excretory capa- the placenta, when the load of bilirubin increases, http://pmj.bmj.com/ city in the course of the maturation of the liver for effective transfer across the placenta to the (Lester & Schmid, 1964). Thus in almost all the maternal circulation the gradient must increase cases where severe haemolysis occurred or con- correspondingly. Thus in cases of severe HDN and tinued at the end or after the 1st week of life (in particularly in the cases maintained alive, in spite of cases of G-6-PD deficiency, pyknocytosis with or very rapid haemolysis, through intrauterine trans- without G-6-PD deficiency and naphthalene inhala- fusions, relatively high serum-bilirubin values exist tion) (Valaes, Doxiadis & Fessas, 1963; and personal in intrauterine life. These infants are known to be on September 27, 2021 by guest. Protected material) high values of conjugated bilirubin were born with high conjugated bilirubin values in the observed with no other evidence of obstruction to cord-blood and go on to show the so-called 'inspiss- the bile flow. Thus it appears that at the end of the ated bile syndrome'. In such a case it was possible 1st week of life and approximately till the end of to estimate the conjugation of bilirubin in the first the 1st month, at least under the influence of high few days of life. It was found that it exceeded, in bilirubin loads (and serum bilirubin concentrations) relation to weight, six times the normal amount the conjugating capacity of the liver increases more excreted by an adult (Valaes, 1963; Maisels et al., rapidly than its excretory ability for bilirubin. 1968). Bakken & Fog (1967a) explained the appearance Thus as noted by Bakken & Fog (1967a), we can of conjugated bilirubin in the serum of normal say that in HDN the high bilirubin values existing newborns on the 2nd to 3rd days and its decrease in intrauterine life trigger the maturation of the after the 6th to 7th days on the same principle of conjugating system and what usually happens after phase difference between the maturation of the the onset of extrauterine life in these infants shifts to conjugating and excretory systems. It should be the prenatal period. Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
88 Timos Valaes Simulating these conditions Bakken & Fog Neonatal jaundice: a problem with many unknowns (1967b) demonstrated that the bilirubin-glucuronyl From the above discussion it follows that in every transferase activity of the liver of newborn rats was case of neonatal jaundice we should consider the increased if the mothers were loaded with bilirubin contribution made by three separate factors: (a) shortly before delivery. These observations production of bilirubin, (b) insufficient conjugatory strengthen the attempts to stimulate the develop- capacity, and (c) inhibition ofconjugation. The inter- ment of the glucuronidation system by substances relationship between these factors can be expressed given to the mother or the newborn infant. Such as follows: substances that gave promising results in animal (a) Degree of hyperbilirubinaemia = bilirubin experiments include 3, 4-benzpyrene (Inscoe & production - bilirubin excretion. Axelrod, 1960); phenobarbitone and nikethamide (b) Bilirubin excretion = conjugating capacity - (Careddu et al., 1964; Kato, Loeb & Gelboin, inhibition of conjugation.* 1965). Evidence is already accumulating that pheno- (c) Degree of hyperbilirubinaemia = bilirubin barbitone is effective in promoting the conjugating production - (conjugatorycapacity - inhibition capacity of the liver in human newborns and thus of conjugation). decreases the degree ofneonatal hyperbilirubinaemia Each one of the three factors can vary indepen- (Crigler & Gold, 1966; Yaffe et al., 1966; Trolle dently of the other two in individual cases and 1968; Khanna et al., 1968; Maurer et al., 1968). moreover the relative importance of each factor can On the other side other hepatic conjugating change rapidly in the first few days of life. At systems such as N-acetyl-transferase have diminished present neither bilirubin production nor conjugating activity during foetal life with no evidence that this capacity and inhibition can be measured accurately might be an advantage or that maturation is sub- on a practical basis. This explains why neonatal strate dependent (Weber & Cohen, 1968). There is jaundice is always a problem with many unknown some evidence that in this enzyme system (Weber & variants even when the main mechanism is known. Cohen, 1968) as well as in glucuronyl transferase, For the sake of simplicity, it has been usual to maturation is connected with a switch from a foetal classify neonatal jaundice by stressing the mostby copyright. form of the enzyme to the adult one (Krasner & important factor in each group of cases. Thus we can Yaffe, 1968). separate the cases of neonatal jaundice in three main In any case the factors that bring about the groups: (a) those in which an increased bilirubin pro- maturation of the enzyme systems are not only of duction is the main factor, (b) those with a much theoretical but also of immense practical interest decreased conjugatory capacity, and (c) those in as the speeding up of the maturation process con- which inhibition of conjugation is the predominant stitutes the simplest solution to most of the thera- factor. This classification is depicted graphically in peutic problems posed by neonatal hyperbili- Fig. 1. It is obvious that within each group differ- rubinaemia. ences in the of between degree hyperbilirubinaemia http://pmj.bmj.com/ the individual cases can be due to variation in each Inhibition ofconjugation of the three factors. This is also true for the so-called In considering the bilirubin-conjugating capacity 'physiological jaundice'. of the newborn in the last few years it became The above, rather complicated, picture should not apparent that another factor ought to be taken into obscure the fact that, had the newborn been endowed account. The serum of women at term as well as with a bilirubin excretory capacity anything near the the serum of the newborn infants contains a sub- adult level, there would not be problems in neonatal stance which can inhibit in an in vitro system the jaundice. It is the absence of reserves in the excretory on September 27, 2021 by guest. Protected conjugation of bilirubin (Lathe & Walker, 1958; capacity that gives importance even to small changes Waters, Dunham & Bowen, 1958; Hsia et al., 1960). in bilirubin production or in inhibition. In that sense This inhibitory effect could be demonstrated in the neonatal period is the most sensitive one for the the breast milk ofsome women whose infants showed clinical manifestation of degrees of haemolysis prolonged mild hyperbilirubinaemia-'breast-milk which in other periods remain clinically silent jaundice' (Katz & Robinson, 1965; Stiehm & Ryan, (Valaes, 1961). 1965; Rosta & Szoke, 1965; Gartner & Arias, 1966). Outside the neonatal period the rate of red-cell Similarly greatly elevated inhibitory activity was destruction must increase two- to three-fold to be of detected in the serum ofsome mothers whose infants any consequence and usually the result is a com- showed marked unexplained hyperbilirubinaemia (Arias et al., 1965). This inhibitory effect in the serum * To simplify and stress the steps of practical importance or the breast milk is connected with in this formula the phase of the excretion of conjugated bili- progestational rubin has been omitted. Similarly the enterohepatic circula- agents (Newman & Gross, 1963; Gartner & Arias, tion of bilirubin, although it might prove of considerable 1964; Arias et al., 1964). importance in the neonatal period, is not considered. Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonataljaundice 89
Production Excretion Production Excretion Inhibition Inhibition Conjugating , t i Conjugating I *capacity capacity Hepatic cell .Hepatic cell (a) (b)
(c) (d) FIG. 1. Basic mechanisms of neonatal jaundice. (a) 'Physiological' jaundice, (b) haemolytic jaundice, (c) jaundice from insufficient conjugation, and (d) jaundice from increased inhibition. pensated haemolytic syndrome with no anaemia or and the membrane from oxidative degradation. jaundice. Under similar conditions in the neonatal These requirements are met through the metabolism period the increase of the bilirubin load two- or of glucose (Fig. 2) which provides both energy and three-fold may result in severe jaundice and kernic- compounds with redox potential. Energy is generated terus. Thus in cases of severe jaundice in the new- mainly in the form of adenosine triphosphate (ATP) born period the concentration of haemoglobin in the Embden-Meyerhof anaerobic glycolytic cannot be used as a criterion of haemolysis. It has pathway and is required to operate the sodium and been estimated, on the basis of the total bilirubin- potassium pump. Reduced nicotinamide adenine by copyright. space and the total mass of haemoglobin of the new- dinucleotide (NADH, or reduced diphosphopyridine born, that an increase of serum bilirubin concentra- nucleotide, DPNH) is an essential co-factor of the tion exceeding 5-6 mg/24 hr cannot be accounted anaerobic pathway and is also responsible for for solely by defective bilirubin excretion and the reduction of methaemoblobin constantly gener- indicates increased haemolysis (Valaes, 1963). ated in the red cell. In Table 1 an aetiological classification of neo- The pentose-phosphate pathway (PPP) of oxida- natal jaundice is attempted excluding conditions tive glycolysis, although under usual condition with an obstructive element and high conjugated involving only approximately 10% of the meta- bilirubin values. From the conditions listed in Table bolism of glucose, is of great importance. This cycle 1 only the ones related to the newly opened field of maintains glutathione in the reduced state through http://pmj.bmj.com/ abnormalities of the red-cell glucose metabolism will the generation of reduced nicotinamide adenine be discussed. dinucleotide phosphate (NADPH, or reduced tri- phosphopyridine nucleotide, TPNH). Under oxidant stress this pathway can be stimulated many-fold the Neonatal jaundice connected with defects in the normal rate and this permits the red cell to maintain pathways of glucose metabolism ofthe red cell its in the of oxidant substances Introduction viability presence either exogenous or endogenous (i.e. ascorbic acid, on September 27, 2021 by guest. Protected The mature red cell is a highly specialized cell cysteine). Reduced glutathione is important for the which has evolved to perform the important function protection of the sulphydryl groups of the haemo- of oxygen transport. It is a cell which, although globin, of the red cell enzymes and of the membrane, endowed with limited metabolic capacities, can fulfil and thus is an all-essential compound for the its purpose with a minimum oxygen consumption survival of the red cell. and energy expenditure. A detailed account of red-cell metabolism is To remain viable the red cell must maintain its beyond the scope of this work and the reader is volume, bi-concave shape and plasticity in the face referred to many recent reviews and monographs of an osmotic and electrochemical gradient and (Prankerd, 1961, 1965; Carson & Tarlov, 1962; constant wear-and-tear while passing through the Harris, 1963; Jandl, 1966; Hoffman, 1966; Carson & narrow capillaries. The red cell needs also to generate Frischer, 1966; Keitt, 1966). Red-cell age is of great an oxidation-reduction potential in order to be importance to metabolism. The pattern of enzyme able to protect haemoglobin from being oxidized to activity and the metabolic rate are closely related to methaemoglobin and to protect the other enzymes the age (Marks, Johnson & Hirschberg, 1958). The Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
90 Timos Valaes TABLE 1. Aetiological classification of neonatal jaundice (of the non-obstructive type) Hyperbilirubinaemia in the newborn from increased bilirubin production (A) With no increase in the rate of haemolysis (1) High total haemoglobin mass (intrauterine hypoxia, syndrome of intrauterine transfusion in uniovular twins, materno- foetal transfusion) (2) Liberation of haemoglobin from extravasated red cells (extensive bruising, giant sub-aponeurotic cephalhaematoma) (B) With increased rate of haemolysis (1) Isoimmunization (Rhesus, ABO, other blood groups) (2) Metabolic defects of the red cells (a) Non-specific (in all newborns and more so in the pre-term) (b) Specific defects in the glycolytic enzymes: (i) Of the anaerobic pathway (Embden-Meyerhof) Pyruvate kinase deficiency Triosephosphate isomerase deficiency 2,3-Diphosphoglycerate mutase deficiency Hexokinase deficiency (Glucosephosphate isomerase deficiency)* (ii) Of the oxidative pentose phosphate pathway Glucose-6-phosphate dehydrogenase deficiency 6-Phosphogluconate dehydrogenase deficiency (Glutathione reductase deficiency)* (c) Other enzyme and metabolite defects Glutathione peroxidase deficiency (Adenosine triphosphatase deficiency)* (Catalase deficiency-acatalasia)* (Congenital absence of reduced glutathione)* (d) Red cell membrane defects Hereditary spherocytosis Elliptocytosis Stomatocytosis
K.K. disease by copyright. (High sodium-low potassium disease)* (e) Haemoglobinopathies Hydrops foetalis in homozygous a-Thalassaemia Congenital haemolytic anaemia with unstable haemoglobins (3) Injury by drugs Anaemia with Heinz-bodies (Vitamin K analogues, naphthalene intoxication, etc.) Infantile pyknocytosis (some cases) (4) Infections Bacterial (E.coli) Viral (cytomegalic inclusions, neonatal hepatitis) Toxoplasmosis
Syphilis http://pmj.bmj.com/ Hyperbilirubinaemia in the newbornfrom deficient conjugating capacity (A) Present in all types of neonataljaundice as a common factor (B) Marked deficiency playing a primary role in the jaundice of: (1) Pre-term infants (2) Congenital hypothyroidism (C) Congenital hereditary deficiency of the enzyme glucuronyl transferase Crigler-Najjar disease Hyperbilirubinaemia in the newborn from inhibition of bilirubin conjugation on September 27, 2021 by guest. Protected (A) Naturally occurring inhibiting substances: (a) In the serum of pregnant women (b) In the breast milk of some women (1) Probably contributes to all types of neonatal jaundice (2) Prolonged jaundice of the breast-fed infants (3) 'Transient familial non-haemolytic neonatal hyperbilirubinaemia' (B) Inhibition due to drugs Novobiocin Miscellaneous and unclassifiable (A) Increased hyperbilirubinaemia in some clinical conditions (a) Infants of diabetic mothers (b) Infants with Down's syndrome (c) Infants with intestinal obstruction (B) Increased hyperbilirubinaemia in some racial or geographical groups * Not yet described as a cause of neonatal jaundice. Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from Neonatal jaundice 91
Glucose
u ^.,^1,:^^^/^ Ii /ATPf r i I - ADP GluV 6-hosphogluconate Glucose -6 -phosphate G se -6-pPhosphoglucon Phosphohexose | G e-pospate NAD 6-Phospholuconic isomerase dehydrogenase (NADPHi dehydrogenase Fructose - 6-phosphate -- -- Ribulose -5- phosphate + CO2 Phosphofructo- (ATP kinase Pentose --- ADPM \ ^^ phosphate Fructose-1,6-diphosphate / pathway Aldolase 1 / ' Dihydroxyacetone phosphate I Glyceraldehyde 3-phosphate Glyceraldehde- NAD Triosephosphate p hspaeehdoens ( iisomerasesomerase phosphate dehydrogenase NADH I) 1,3-Diphosphoglycerate - - 2,3- Diphosphoglycerate Diphosphoglycerate mutase/ Q>c-Q Phosphoglycerate | .ADP 2,3- Diphosphoglycerate phosphatase kinase t ATP/ Inorganic phosphate by copyright. 3-Phosphoglycerate Triose mutase E 2-Phosphoglycerate W Enolase $ Phosphoenolpyruvate I ADP Pyruvate kinase T A\ATP Pyruvate NADH Lactic dehydro- I(N http://pmj.bmj.com/ genase NAD Lactic acid FIG. 2. Metabolism of glucose. failure of the red cell to maintain its energy require- erythrocytes in the newborn period can be explained ments, through degradation of rate-limiting enzymes on the basis of younger mean age of the red-cell of the glycolytic pathway, is the cause of its limited population including increased enzyme activity on September 27, 2021 by guest. Protected life-span. Studying a metabolic parameter in a blood (hexokinase, glyceraldehyde-3-phosphate de- sample we only measure the mean value of a hydrogenase, pyruvate kinase, glucose - 6 - phos- heterogeneous population which includes all the phate dehydrogenase, 6-phosphogluconic dehydro- range between the very young and the very old cells. genase), increased glucose consumption and in- Sometimes measurements of enzyme activity, or creased ATP content (Gross et al., 1963). The even more important, clinical manifestations can be shortened life-span of the red cells of the newborn, altered by a change in the mean red-cell age. An increased mechanical fragility, increased vulner- example of this can be found in the newborn period. ability to oxidative stress with methaemoglobin As a result of the shortened life-span of foetal red accumulation and denaturation of haemoglobin in cells and the rapid expansion of total red-cell mass the form of Heinz-bodies and greater tendency to during the rapid growth of the last trimester of form 'spickled' red-cell forms on incubation, pregnancy the mean red-cell age at birth is much demonstrate the metabolically precarious state of lower. Some of the metabolic peculiarities of the the erythrocytes in the newborn period (Oski and Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
92 Timos Valaes Naiman, 1965; Oski, 1967). Marks, Szeinberg & Banks, 1961; Kirkman, 1962) The normal activity and response to stimulation have proved that the variants so far studied were the of the PPP of the erythrocytes in the newborn result of a structural mutation leading to differences period indicates that failure of this pathway is not from the normal enzyme in one or more of its func- responsible for their vulnerability to oxidant stress tional characteristics: rate of decay, specific activity (Oski, 1967). The low activity of the erythrocytes of per molecule of enzyme, substrate affinity and the newborn in catalase, glutathione peroxidase and substrate specificity. In this respect it should be NAD-dependent methaemoglobin reductase (dia- mentioned that assays of enzyme activities in vitro do phorase is a likely explanation for this vulnerability not represent the conditions in vivo. The conditions (Bracci et al., 1965; Gross et al., 1967). The erythro- of the assays are those of optimum substrate con- cytes of the newborn are also characterized by centration and the activity is measured in the whole glutathione instability (Zinkham, 1959; Oski & population of the red cells. Thus diminished affinity Naiman, 1965), ATP-increase without relative in- of the enzyme for the substrate or a speeding up of crease in ADP (Gross et al., 1963) and ATP- enzyme decay in older cells escape recognition. instability. Special studies are required for the detection of variants which produce such alterations in the General remarks on red cell enzymopathies behaviour of an enzyme. Moreover structural vari- Enzyme variants leading to defective enzyme ants of the enzyme molecule might be of no func- activity have been described for almost all the tional significance and only their electrophoretic or enzymes involved in the two pathways of glucose chromatographic behaviour distinguishes them from metabolism of the erythrocyte. These enzymo- the normal. pathies have manifested clinically either as con- Autosomal recessive transmission has been proved genital non-spherocytic haemolytic anaemia (CNHA) for most of the enzyme defects. The homozygous or as haemolysis following exposure to exogenous individuals are affected clinically while the hetero- agents, particularly primaquine and fava beans and zygous carriers are asymptomatic although there as finally severe neonatal jaundice. The drug-induced are a few exceptions to this rule. For most of the by copyright. haemolysis is connected with defective function of enzyme studies it appears that different loci are the PPP, while CNHA can result from defects in directing the formation of the same enzyme in either the Embden-Meyerhof pathway or PPP. different tissues. Thus in red-cell pyruvate kinase The defects which produce CNHA have one thing and hexokinase deficiency the white-cell enzymes in common, they are very rare. This is not a false are normal. G-6-PD differs from other enzymes in impression created by the relatively recent develop- being sex-linked (Childs et al., 1958) and the same ment of the laboratory methods for their detection. locus is responsible for the enzyme in probably all The clinical condition described as CNHA (Dacie et the tissues (Nitowsky et al., 1965; Lunder & al., 1953) is itself very rare (de Gruchy et al., 1960). Gartler, 1965; Chan, Todd & Wong, 1965; Justice The importance of these rare defects lies in the fact et al., 1966; Yoshida, 1968). http://pmj.bmj.com/ that through the study ofsuch 'experiments ofnature' In relation to neonatal jaundice red cell enzymo- our knowledge ofred-cell metabolism is enlarged. pathies are of interest as severe hyperbilirubinaemia Glucose-6-phosphate dehydrogenase (G-6-PD) might be the first manifestation of CNHA and deficiency, the first enzyme defect to be described G-6-PD deficiency may manifest in the neonatal (Carson et al., 1956) is much commoner. It has been period with severe jaundice in some population estimated that over 100 million people are affected groups. The whole subject has been recently reviewed (Carson, 1960). In many populations high fre- (Oski, 1965). quencies of the G-6-PD deficiency gene are prevail- on September 27, 2021 by guest. Protected ing thus constituting an important genetic poly- Enzyme defects of the anaerobic glycolytic pathway morphism (Motulsky & Campbell-Kraut, 1961; (Embden-Meyerhof) Marks, 1963; Motulsky, 1963). Glucose-6-PD is the The anaerobic pathway of glycolysis is the main only enzyme studied so far in detail both in its supplier of energy for the red cells and defective normal type and the abnormal variants. It can serve operation of this pathway greatly limits their life- as a prototype for our understanding ofthe abnormal span. Accordingly in most of the cases severe jaun- variants of other red-cell enzymes. The extensive dice was expected to occur in the neonatal period. studies of Yoshida and collaborators (Yoshida, 1966, Actually, in only a small proportion of the cases so 1968; Yoshida, Stamatoyannopoulos & Motulsky, far reported was neonatal jaundice conspicuous, 1967) as well as previous studies (Carson, Schrier & while anaemia was obvious very early in infancy. It Kellermeyer, 1959; Kirkman, Riley & Crowell, is possible that although an increased rate of haemo- 1960; Kirkman & Hendrickson, 1963; Kirkman, lysis exists in the neonatal and intrauterine periods, Schettini & Pickard, 1964a; Kirkman et al., 1964b; these infants do not develop severe jaundice because Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonatal jaundice 93 TABLE 2. Pyruvate kinase deficiency with manifestation in the neonatal period Cases with Onset of Ethnic Total icterus Exchange anaemia in References origin cases neonatorum transfusion infancy
Six North European, one Mexican 7 2 1 4 Tanaka, Valentine & Miwa (1962) North European 3 2 1 2 Oski & Diamond (1963) French 1 I Boivin & Mallarm6 (1963) Italian 2 ? ? 2 Brunetti et al. (1963) German 1 1(+ 2 siblings) I 1 Busch (1963) and Busch et al. (1966) English 1 1(Rhesus?) 1 1 Bowdler & Prankerd (1964) North European 1 1 l(Kernicterus?) 1 Oski et al. (1964) Italian 1 ? - 1 Bestetti et al. (1964) Amish 21 6+ 3(1 Kernicterus) 21 Bowmann et al. (1965) North European 2 2(+ 1 sibling) -2 Keitt (1966) Italian 1 1(+ 1 sibling) 1(+ 1 sibling) I Volpato et al. (1968) Total 41 16 9 37 ofbetter thanaverage bilirubin conjugationandexcre- described by Bowmann, McKusick & Drowamrazu tion. Further discussion on this point must wait for (1965). More detailed study of the enzyme in the more clinical and laboratory details on the natural affected individuals is required in order to see course during the neonatal period of enzymopathies whether different pathological variants are involved. of the anaerobic pathway. Differences in the enzyme structure, kinetics and stability similar to those described for G-6-PD may Pyruvate kinase (PK) deficiency is after G-6-PD account for the clinical variability. Already CNHA deficiency the commonest enzyme defect found in has been described with normal PK activity, with by copyright. cases of CNHA. More than eighty cases have been the usual assay conditions, but with very much reported so far (for list of references see Keitt, diminished substrate affinity (high Km constant) of 1966; Volpato, Vige & Cattarozi, 1968). It is the enzyme (Paglia et al., 1967; Sachs et al., 1967; impossible to estimate the percentage of cases with Zarkowsky et al., 1968; Miller et al., 1968). The lack neonatal manifestations, as in many of the reports no of uniformity in the metabolic lesion as well as the clinical details are given, or vague descriptions such difficulty in explaining the clinical severity on the as 'anaemia or icterus present since birth' make it basis of abnormalities in the glycolytic energy difficult to form a clearcut picture of the neonatal metabolism of the red cell has been discussed course in this enzymomopathy. In Table 2 we have thoroughly by Keitt (1966). listed all the reports which include cases of PK http://pmj.bmj.com/ deficiency with neonatal manifestations. Triosephosphate isomerase deficiency has been In approximately half of the cases severe neonatal found in members of three families with CNHA. jaundice and anaemia developed in the first few days Two of the families are related and consanguinity oflife. In a large proportion ofthese infants exchange exists in one of them. The common racial origin of transfusion was performed while kernicterus the other family makes it possible that all three occurred in two infants. Generally speaking, in this families have a common ancestry. Five children of defect individuals with these families were and four of them enzyme homozygous present homozygotes on September 27, 2021 by guest. Protected chronic haemolysis often with exacerbations in the presented severe anaemia in the 1st month of life course of infections, while their heterozygous parents but no neonatal jaundice, while the other one had or relatives are asymptomatic. Nevertheless, in at severe enough jaundice to require exchange trans- least three of the cases with early onset of severe fusion. Two more infants died early, one during haemolysis partial PK deficiency was present (Oski exchange transfusion and the other on the 6th day & Diamond, 1963; Busch et al., 1966; Volpato et from anaemia (Schneider et al., 1965; Shore, al., 1968). This is only one example of the poor Schneider & Valentine, 1965; Valentine et al., 1966). correlation between severity of clinical manifesta- Progressive neurological disease and frequent tions and the deficiency in enzyme activity as bacterial infections with unexpected sudden death assessed in vitro. The severity ofmanifestations seems was the characteristic of the clinical histories in those to be more uniform if members of the same family surviving the neonatal period. This clinical picture are considered (Busch et al., 1966; Keitt, 1966; suggested multiple tissue involvement. Actually the Volpato et al., 1968) or the siblings are all of enzyme activity of white blood cells, muscle, common origin as are the cases in the Amish isolate cerebrospinal fluid and serum was found to be low Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
94 Timos Valaes in such patients (Schneider et al., 1968). picture. Heterozygotes with 50% enzyme activity have been described as asymptomatic (Brewer & 2,3-Diphosphoglycerate mutase deficiency was Dern, 1964) but one patient had CNHA with no almost certainly the cause of severe anaemia at evidence of susceptibility to drug-induced haemoly- birth, but no jaundice, in an infant which demon- sis (Scialom, Najean & Bernard, 1966). Finally one strated a severe haemolytic process for the duration male infant with 6-PGD deficiency had jaundice and of his life (he died from bronchopneumonia at 7 anaemia in the first hours of life and an exchange weeks). The parents were first cousins and the transfusion was performed (Lausecker et al., 1965). enzyme activity in them was 50% of the normal At the age of 2-3 months this infant continued to (Schr6ter, 1965). In two more cases with neonatal have anaemia and slight jaundice. manifestations this enzyme defect was inferred from the low levels of 2,3-diphosphoglycerate (Lohr & Glutathione reductase deficiency (GSSG-R) with Waller, 1963; Lelong, Alagille & Odievre, 1964). values of enzyme activity ranging from 16 to 50% The clinical picture was completely different from of normal has been connected either with sus- the case of Schr6ter (1965) and we must assume ceptibility to drug haemolysis (Carson, Brewer & either a different genetic variant or a defect in an Ickes, 1961; Carson et al., 1963) or with CNHA enzyme proximal to 2,3-DPG mutase. (Lohr & Waller, 1962; Waller et al., 1964, 1965). So far neonatal jaundice has not been associated Hexokinase deficiency was found in a girl with with GSSG-R deficiency. severe anaemia and jaundice appearing in the first Glutathione peroxidase deficiency (GSH-P) of the few hours of life and necessitated an exchange red cells has been connected with severe neonatal transfusion at the age of 4 hr (Valentine et al., jaundice and anaemia in the newborn period 1967). The haemolytic process continued after the (Necheles, Boles & Allen, 1968). Four cases have neonatal period and frequent transfusions were been described in Causasian newborns. Two of these required up to the time of splenectomy, following infants were born pre-term. There was no history of which she improved, although the haemolytic state exposure to haemolytic agents. The haemolysis was by copyright. remained. The critical position of hexokinase in the fairly severe and one of the infants was treated by glucose metabolism of the red cell explains the exchange transfusion. Nevertheless all the infants severity of the haemolytic process in this case. were asymptomatic 3 months later and in this Glucose phosphate isomerase deficiency has been respect their course is similar with that of the described in two families with CNHA. No clinical G-6-PD deficient infants who develop severe details have been published so far (Baughann et al., jaundice. In vitro, the red cells demonstrated Heinz- Holland et body formation on incubation with acetylphenyl- 1967; al., 1968). hydrazine. From genetic evidence it was inferred that the infants were as one of the Enzyme defects of the pentose phosphate pathway heterozygotes only http://pmj.bmj.com/ (PPP) other than glucose-6-phosphate dehydrogenase parents had diminished enzyme activity. These deficiency. cases demonstrated the precarious state of the red With the enzymes of PPP, glutathione reductase cells of the newborn in the face of endogenous and peroxidase deficiency and congenital deficiency peroxidation. Low catalase and GSH-P activity in glutathione will be examined as they are part of characterizes the red calls of the newborn and is the same defensive mechanism of the erythrocyte one of the mechanisms for their increased sensitivity against oxidative degradation. The importance of to oxidative drugs. Obviously the further depression these mechanisms for the survival ofthe red cell under of GSH-P activity due to a genetic defect explains on September 27, 2021 by guest. Protected conditions of oxidative stress have been already the severity of haemolysis during the neonatal mentioned. When this pathway cannot respond to period even in heterozygotes. such a stress an haemolytic crisis with red-cell Congenital absence of reduced glutathione (GSH) fragmentation and Heinz-body formation results. has also been connected with susceptibility to drug A minimum activity of PPP is required for the haemolysis but no reference of neonatal jaundice is survival of the red cell under normal conditions found in the few cases reported (Oort, Loos & even in the absence of any exogenous oxidative Prins, 1961; Prins et al., 1966; Waller & Gerok, assault. If these minimum requirements cannot be 1964; Boivin & Galland, 1965; Prins, Loos & met, chronic haemolysis results with the clinical Zurcher, 1968). picture of CNHA. Glycose-6-phosphate dehydrogenase deficiency 6-Phosphogluconate dehydrogenase deficiency (6- Glycose-6-phosphate dehydrogenase deficiency PGD) is not connected with a uniform clinical was discovered in the course of studies for the eluci- Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonatal jaundice 95 TABLE 3. Common variants of glucose-6-phosphate dehydrogenase Activity in Substrate Variant red cells Race* Clinical Substrate analogue Basic effect (%) manifestations affinity utilization of mutation Normal (B+) 100 All None - - Normal fast (A+) 88 Negro None Normal Normal Structural mutation, Aspara- gine replaced by aspartic acid in enzyme molecule Negro type of de- Drug induced Structural mutation. Accelera- ficiency (A-) 10-20 Negro haemolysis Normal Normal ted degradation but normal self limited synthesis (normal activity in young RBCs and WBCs) Mediterranean 0-7 Greeks, Italians Drug induced Increased Increased Structural mutation leading to type of deficiency Sephardic haemolysis. less activity per molecule and (B-) Jews, Indians Favism. Neo- accelerated degradation natal jaundice (young RBCs and WBCs have diminished activity) Canton 4-24 South Chinese Drug induced Increased Slightly haemolysis. Neo- Increased ? natal jaundice * In many populations with high incidence of G-6-PD deficiency the variant has not been fully characterized. dation of the mechanism of the haemolytic effect of The main characteristics of the common variants primaquine (Dern, Beutler & Alving, 1954; Carson of G-6-PD are shown in Table 3. The A+ variant et al., 1959; Beutler, 1966). The development of found in approximately 18% of the Negroes has no relatively easy screening tests (Motulsky & Campbell- functional differences from the normal enzyme but by copyright. Kraut, 1961; Fairbanks & Beutler, 1962; Brewer etal., moves faster in electrophoresis. Fingerprinting 1960; Bernstein, 1962) has permitted extensive popu- proved that it results from a single amino acid lation surveys. A high incidence has been found in substitution, asparagine being replaced by aspartic populations around the Mediterranean, in Negroes, acid (Yoshida, 1968). For the Negro type of G-6-PD in many population groups of South Asia and among deficiency (A-) a structural mutation is again Chinese. On the basis of its geographical distribu- suggested from its different behaviour on Sephadex tion, which coincides with that of endemic malaria, a column. The enzyme has normal activity per molecule selective advantage of G-6-PD deficiency in malar- but both in vitro and in vivo is easily and irreversibly ious areas was assumed (Motulsky, 1960, 1964). inactivated. Young erythrocytes and nucleated cells Extensive enzymological studies have revealed the (for instance white cells) which can synthesize new http://pmj.bmj.com/ heterogeneity of G-6-PD deficiency as well as enzyme molecules, have normal enzyme activity. variants of the enzyme with no functional conse- The overall effect of this variant can be described as quences (Carson & Frischer, 1966; Kirkman, accelerated ageing of the red cells. These charac- Kidson & Kennedy, 1968; Yoshida, Stamatoyan- teristics explain the self-limited course ofprimaquine- nopoulos & Motulsky, 1968). induced haemolysis in the Negro type of G-6-PD A molecular weight of 240,000 and a composition deficiency (Kellermeyer et al., 1961). from six sub-units of molecular weight 40,000 has In the Mediterranean type of G-6-PD deficiency been suggested from the characteristics of the (B-) the enzyme activity is almost zero and even the on September 27, 2021 by guest. Protected purified and crystallized normal enzyme (Yoshida, young cells and reticulocytes have greatly diminished 1966). Over forty variants of G-6-PD have been activity (one-third of normal). The decay of the characterized so far. The distinction is based on enzyme is also accelerated. A structural mutation enzyme activity in the erythrocytes and other cells, is again suggested leading to disturbance of the electrophoretic and column chromatography mobil- secondary and tertiary configuration of the enzyme ity, affinity for the main substrates (G-6-P and molecule and thus to diminished activity per enzyme NADP) and for substrate analogues (2-desoxy- molecule and accelerated degradation (Yoshida et glycose-6-phosphateandgalactose-6-phosphate),heat al., 1968). These characteristics explain: (a) the lability, pH optimum, neutralization with specific more severe haemolytic effect of primaquine and the antisera, estimation of enzyme activity in red cell absence of a refractory period in subjects with this precursors and young and old erythrocytes and type of G-6-PD deficiency (Salvidio et al., 1967; finally on clinical manifestations and racial distribu- George et al., 1967). (b) The fact that the list of tion. drugs that produce haemolysis is more extensive Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
96 Timos Valaes than that for the Negro-type of G-6-PD deficiency in the red cell precursors while in the Negro and and includes also four beans. Mediterranean type of G-6-PD deficiency the activity All G-6-PD variants are alleles at the same locus is normal in the precursors but declines rapidly of the X chromosome (sex-linked). The sex-linkage because of in vivo instability. of the enzyme defect results in the existence of two In several cases of CNHA from G-6-PD deficiency types of males, i.e. normal and hemizygote defec- severe neonatal jaundice and anaemia were pro- tive; and three types of females, i.e. normal homo- minent (Newton & Bass, 1958; Newton & Frajola, zygotes, defective homozygotes and heterozygotes 1958; Shahidi & Diamond, 1959; Zinkham & with one X chromosome carrying the normal gene Lenhard, 1959; Kirkman & Riley, 1961; Tada, 1961; and the other X carrying the gene for the abnormal Bernard et al., 1963; Greenberg & Tanaka, 1965) variant. In the case of the heterozygotes the red-cell and actually it was in these cases that the association population should be considered a mosaic according between severe neonatal jaundice and a red cell to the Lyon-effect with a proportion of the red cell enzyme defect was first demonstrated. The connec- population derived from normoblasts with active tion between G-6-PD deficiency and severe neonatal X chromosomes with the normal genes (normal jaundice will be examined in detail in the discussion enzyme activity) and the remaining red cells from that follows. normoblasts with active X chromosomes with abnormal genes (decreased enzyme activity). This Severe jaundice in G-6-PD deficient neonates follow- situation explains the difficulties in detecting the ing exposure to haemolytic agents heterozygote females with laboratory methods based The association between G-6-PD deficiency and on the enzyme activity of the whole red-cell popula- severe neonatal jaundice has been reported from tion. The level of enzyme activity, within the normal almost every ethnic group in which this enzymopathy range, seems to be controlled by normal alleles is known to occur. In many of these reports an (isoalleles) and thus is also genetically determined exogenous haemolytic agent could be implicated. (Davidson, Childs & Siniscalco, 1964; Motulsky & The infant was either exposed to the substance or the circulation or Stamatoyannopoulos, 1966). directly through placental by copyright. The metabolic consequences of the primary defect the breast milk. The list of substances implicated in in G-6-PD activity and their relation to drug- the production of haemolysis and severe jaundice in induced haemolysis have been extensively studied susceptible newborns includes but there are still some points poorly understood (a) Naphthalene (moth balls) usually inhaled but (Beutler, 1966; Carson & Frischer, 1966). Several also absorbed through the skin or from breast milk mutants of G-6-PD cause chronic haemolysis in the after ingestion by the mother (Zinkham & Childs, absence of exogenous haemolytic agents. In such 1957, 1958; Dawson, Thayer & Desforges, 1958; cases exacerbation of haemolysis occurs during Valaes, Fessas & Doxiadis, 1961; Valaes et al., 1963; infection or administration of haemolytic drugs. Jim & Chu, 1963; Naiman & Kosoy, 1964).
Although fairly common among the enzymopathies (b) Long acting sulphonamide given to the mother http://pmj.bmj.com/ causing CNHA these variants remain extremely (Brown & Cevik, 1965). rare in comparison with the common mutants of (c) Acetylsalicylic acid and phenacetin (Lee et al., G-6-PD deficiency which are associated with drug- 1961; Harley & Robin, 1962, 1963). induced haemolysis. (d) 'Triple dye', a local antiseptic applied to the The variants connected with CNHA have been umbilical stump (Freier et al., 1965). found mainly in Caucasians of North European (e) Combinations of agents (Ifekwunigwe & origin but there is no reason to believe that such Luzzato, 1966). mutations have not occurred in other racial groups. The group with naphthalene inhalation is the on September 27, 2021 by guest. Protected The characterization of these variants with a variety largest. In many of the cases the circumstances of of enzymological techniques showed that they differ the exposure suggest that only a minute amount of from the normal and the other common variants in the substance could have been absorbed by the having, decreased thermal stability (Kirkman et al., infant, yet the ensuing haemolysis was dramatic 1964b; Beutler, Mathan & Smith, 1968), decreased with an abrupt drop in the haemoglobin concentra- affinity for G-6-P or/and NADP (Pinto et al., 1966; tion and occasionally with haemoglobinuria and Beutler et al., 1968) and differences in the pH opti- methaemoglobinuria. Fragmentation and Heinz- mum (Beutler, 1968). Some other variants have not body formation in a large proportion of the erythro- as yet been characterized in detail. The abnormal cytes was also noted in many cases (Valaes et al., stability and kinetics of the enzyme variants explain 1963). The level of bilirubin was related not only to the severity of haemolysis. Recently defective syn- the degree of haemolysis but also to the age of the thesis of the enzyme has been described (Piomelli infant. The earlier the exposure to the haemolytic et al., 1968). The enzyme was found to be absent even agent, naphthalene or other, the higher the level of Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonatal jaundice 97 bilirubin tended to rise. In many cases kernicterus among Sardinian (Panizon, 1960a,b), Chinese occurred even before an appreciable anaemia had (Smith & Vella, 1960; Yue & Strickland, 1965; Lu, time to develop (Valaes et al., 1961, 1963; Naiman Wei & Blackwell, 1966; Wong, 1964, 1966; Brown & & Kosoy, 1964). It should be noted that a similar, Wong, 1968), Greek (Doxiadis, Fessas & Valaes, but somewhat milder, haemolytic effect of naphtha- 1960; Fessas, Doxiadis & Valaes, 1962; Zannos- lene was seen in a few infants with no demonstrable Mariolea et al., 1968), Thai (Flatz et al., 1963), enzyme defect (Valaes et al., 1963). Malay (Wong, 1966) and Turkish (Say et al., 1965) Vitamin K analogues, in large doses, have been newborns. In these ethnic groups G-6-PD deficiency connected with hyperbilirubinaemia and kernicterus was one of the major causes of severe jaundice and particularly in premature newborns (Gasser, 1953; even more the major cause of kernicterus. An Allison, 1955; Lawrence, 1955; Crosse, Meyer & exogenous haemolytic agent could not be demon- Gerrard, 1955; Meyer & Angus, 1956; Bound & strated in the majority of the infants. In the case of Telfer, 1956; Lucey & Dolan, 1959). Overt haemo- Chinese newborns traditional herbs used in the care lysis and evidence of a toxic effect, as expressed by of mother and baby were blamed. As nothing mys- the presence of Heinz-bodies in the erythrocytes terious or exceptional was used in the conduct of of many of the reported cases, clearly demonstrate labour and the care of the mothers and infants in that vitamin K analogues can stress the limited most of the Greek and Italian cases, it was claimed resources of anti-oxidant mechanisms of the new- that severe jaundice could develop in G-6-PD born. In vitro it was shown that incubation of the deficient newborns in the absence of an exogenous red cells of the newborn with menandione sodium haemolytic agent. bisulphite lowered the glutathione content similarly The main clinical characteristics of this type of to acetylphenylhydrazine (Zinkham, 1959). The jaundice (Doxiadis & Valaes, 1964) are as follows: vulnerability of the G-6-PD deficient red cells to (a) In the majority of the cases (70-75 %) the onset oxidants makes it likely that vitamin K analogues of jaundice occurs between the 2nd and 3rd day of will also be damaging to them. Among Greek infants life. In a small proportion (10-15%) of the infants with severe jaundice connected with G-6-PD jaundice is noted within the first 24 hr of life. Onset by copyright. deficiency those who received these analogues, even on the 4th or up to the 8th day of life has also been in small doses, presented with a much higher per- recorded. No correlation seems to exist between the centage of kernicterus than the remainder. In many time of the onset of the jaundice and the severity of these infants fragmented erythrocytes, Heinz-body of subsequent hyperbilirubinaemia. From the formation and low haemoglobin values formed a clinical point of view it should be stressed that in clearcut picture of haemolytic crisis (Doxiadis et al., most cases the time of onset of the jaundice is 1961; Valaes et al., 1961; Doxiadis & Valaes, 1964). similar to that of the so-called 'physiological It has to be admitted that in most cases the analogues jaundice' although the course and outcome are quite were given after the onset of the jaundice. different. In some infants the late onset of jaundice Two controlled trials, in Negro populations, and continuous rise of serum bilirubin well into the http://pmj.bmj.com/ failed to show any effect of even large doses of 2nd week of life, made it possible for kernicterus to vitamin K analogues on the serum bilirubin levels develop after the infants had left the maternity of G-6-PD deficient newborns (Capps et al., 1963; ward. This explains why, in areas where severe Zinkham, 1963). In the study of Zinkham it was neonatal hyperbilirubinaemia from other causes is suggested that natural vitamin K lowered the levels effectively managed, kernicterus still occurs in of serum bilirubin. G-6-PD deficient newborns. Thus at present there is circumstantial evidence Kernicterus can occur or the for (b) necessity on September 27, 2021 by guest. Protected that in newborns with the more severe, Mediter- exchange transfusion arise as early as the 2nd day of ranean, type of G-6-PD deficiency vitamin K life and as late as the 11th day. In 65 % of the cases analogues may have an haemolytic effect, even in the decisive point in the course of hyperbilirubinae- small doses, while controlled experiments demon- mia is reached on the 3rd, 4th or 5th day of life. strated the absence of such an effect in newborns (c) The jaundice is self-limited although pro- with the Negro type of G-6-PD deficiency. tracted in its course. Unless kernicterus supervenes, prognosis is good as there is no evidence of con- Spontaneous severe jaundice in G-6-PD deficient tinuing haemolysis outside the neonatal period newborns (except under the influence of an exogenous haemo- In a number of surveys the incidence of G-6-PD lytic agent). In this respect there is no resemblance to deficiency in groups of infants with severe jaundice the sporadic cases of G-6-PD deficiency connected and kernicterus was examined. In a high proportion with CNHA. of infants, with otherwise unexplained severe (d) The proportion of the red-cell population jaundice, G-6-PD deficiency was demonstrated haemolysed in the course of the jaundice varies from Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
98 Timos Valaes
one case to the other. Usually a small proportion differences in the incidence of severe jaundice among of the red cells is destroyed and no frank anaemia the G-6-PD deficient newborns in the various develops. In other cases the destruction affects almost surveys. all the red cells. Extreme anaemia develops in a few The results of the surveys listed in Table 4 suggest days and most of the red cells in the peripheral blood that there are three types of populations in relation are fragmented and contain Heinz-bodies. In to the incidence of hyperbilirubinaemia in G-6-PD accordance with these clinical observations the few deficient male newborns: (1) Populations with the cases which have been studied with autotransfusion severe type of G-6-PD deficiency (Mediterranean showed 51Cr half-lives varying from 8 to 22 days type) and an incidence of severe neonatal jaundice (personal material; Schettini et al., 1963). of 5-10%. This group includes the Greeks of the (e) Females, proved to be heterozygotes on Southern part of Greece and of Rhodes and also the genetic grounds (i.e. G-6-PD deficient father) and Chinese and Thais of the Bangkok region. (2) with enzyme activity of the red-cell population Populations with the severe type of G-6-PD defici- within the normal range, can exhibit equally severe ency and an incidence of severe neonatal jaundice of degrees of jaundice. In these cases the haemolysis 25-45 %. This group includes the Greeks of Lesbos, of a large proportion of the red cells of the G-6-PD the Chinese of Hong-Kong, Formosa and Singapore, deficient clone is sufficient to produce severe and probably the Turks of the region of Ankara. (3) hyperbilirubinaemia. The male/female ratio in large Populations with the milder, Negro-type, of G-6-PD series of infants with severe neonatal jaundice from deficiency and with very little associated severe G-6-PD deficiency has varied from 3-0 (Doxiadis & neonatal jaundice. Obviously there must be several Valaes, 1964) to 1-5 (Zannos-Mariolea et al., 1968.) other populations belonging to one of the above The smaller ratio was obtained by using more categories that have not yet been investigated in this sensitive laboratory methods for the detection of manner. A retrospective study by Szeinberg and his female heterozygotes. associates (Szeinberg et al., 1963) among different (f) The cases of severe jaundice from G-6-PD communities of non-Ashkenazi Jews with extremely
deficiency are not equally distributed in the whole high frequencies of G-6-PD deficiency of the severeby copyright. group of G-6-PD deficient newborns. There is a type, failed to show any relation between G-6-PD strong familiar predisposition (Panizon, 1960b; deficiency and severe jaundice. Only among Iraqi Weatheral, 1960) which can best be explained by Jews such a relation seemed to exist. the hypothesis that a second independently trans- The incidence of severe jaundice among hetero- mitted hereditary factor is necessary for the develop- zygote female newborns has not been studied in all ment of severe jaundice in G-6-PD deficient neonates the surveys. The screening procedures employed (Fessas et al., 1962). for the detection of G-6-PD deficiency only in the hands of Flatz and his associates (Flatz et al., 1963, Surveys of the incidence of neonatal jaundice among 1964) succeeded in detecting most ofthe heterozygote G-6-PD deficient newborns female newborns (as judged from the comparison http://pmj.bmj.com/ In Table 4 we have tabulated the results of all the between the number found and that expected from available surveys, in which the relationship of the gene frequency, i.e. frequency of male hemi- G-6-PD deficiency and neonatal hyperbilirubinaemia zygotes). In the population of Rhodes, Valaes and have been examined in consecutive series of new- his associates (Valaes et al., 1968) collected family borns. Because of differences in the criteria of severe data and estimated quantitatively the activity of jaundice and the general planning of the surveys G-6-PD in all the females with severe jaundice. The these cannot be considered to be com- number found to be for G-6-PD strictly heterozygotes on September 27, 2021 by guest. Protected parable. For example in the Greek surveys of the deficiency was referred to the expected number in populations of Southern Greece and of Lesbos, the the whole population studied. In this population an surveys in the regions of Bangkok, Hong-Kong and almost equal number of female newborns had Ibadan, serum bilirubin was determined only in severe jaundice as male newborns but in the latter those infants judged to have moderate or marked the jaundice was much more severe and kernicterus jaundice, those with serum bilirubin values of 15-16 occurred only in them. mg/100 ml or over were included in the group with The question of exogenous haemolytic agents severe jaundice. In the surveys at Rhodes (Greece), being the cause of severe jaundice in all the G-6-PD Formosa and among Negroes in U.S.A., the deficient newborns with this manifestation could be bilirubin was determined in all infants with G-6-PD easily ruled out in the above surveys. No relation deficiency and a group of controls. could be found between the usual drugs used during The differences in the methods and criteria and labour and the development of severe jaundice and possibly differences in the bilirubin values between nothing mysterious was used in the care of mother the laboratories involved, cannot account for the and baby. Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
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100 Timos Valaes Cord blood findings in infants with G-6-PD infants and six infants required exchange transfusion. deficiency of the Mediterranean type have been In comparison with controls the deficient premature studied in the island of Rhodes, Greece (Valaes et al., infants had a larger drop in haemoglobin values and 1968) and in Singapore (Brown & Wong, 1968). In higher reticulocyte counts. This increased vulner- comparison with a control group, male infants ability of the premature G-6-PD deficient infants is with G-6-PD deficiency had a significantly lower probably the result of both their decreased bilirubin- mean haemoglobin concentration and higher serum conjugating capacity and their precarious red-cell bilirubin one, but there was no significant difference metabolism which in the presence of G-6-PD in the reticulocyte count. The lower mean haemo- deficiency leads to rates of haemolysis greater than globin concentration was the result of a shift the ones existing in term G-6-PD deficient newborns. towards lower values of the whole distribution Another important fact becomes apparent from the curve of cord blood haemoglobin and was not due data in Table 4. A correlation exists between the to a group of G-6-PD deficient infants with very low incidence of severe jaundice in the G-6-PD deficient values. In the 'Rhodes' survey the serum bilirubin newborns and the incidence, among the normal concentration on the 4th day was 7-88 i 4-8 in the newborns of the same population, of severe jaundice control group and 9-86 ± 6-6 in infants with G-6-PD without foeto-maternal blood group incompatibility. deficiency, full term and with no blood group Thus in Lesbos unexplained severe jaundice was incompatibility. In the 'Singapore' survey the maxi- much more frequent than in either the population of mum serum bilirubin for the Chinese newborn Rhodes, Southern Greece or the Bangkok region. It infants was 11-2 ± 3-7 in the controls and 15-5±3-4 seems that in certain regions or populations an in the G-6-PD deficient infants. The above in com- icterogenic factor exists, which alone is capable of bination with the findings in the cord blood and producing severe jaundice in some infants. In the lower haemoglobin values on the 4th day clearly same populations the combination of this unknown demonstrate that the G-6-PD deficient newborns as factor and G-6-PD deficiency leads to the develop- a group, have increased rates of haemolysis starting ment of severe jaundice in a large proportion of the from the intrauterine life and continuing after birth infants of the latter group (Valaes & Karaklis, 1967;by copyright. leading to higher degrees of neonatal hyperbili- Brown & Wong, 1968). rubinaemia in comparison with infants with normal The presence of an icterogenic factor was demon- G-6-PD activity. In a similar study in Formosa the strated for the first time in Lesbos and was related higher mean serum bilirubin value of the G-6-PD to the high incidence of severe jaundice both deficient infants, in comparison with a control group, among the G-6-PD deficient and the normal infants was obvious even at the first 24 hr of life. This again (Doxiadis et al., 1964). In a later study the level of points to an early, intrauterine, onset of haemolysis serum bilirubin in male full-term infants without (Lu et al., 1966). In the 'Rhodes' survey it was not foeto-maternal incompatibility and with normal clear whether the infants with extreme degrees of G-6-PD activity from Lesbos was compared with a hyperbilirubinaemia (including three cases of kernic- similar group from Rhodes (Valaes et al., 1968). The http://pmj.bmj.com/ terus) were the upper end of a continuous spectrum Lesbos group had significantly higher serum bili- of severity or constituted a separate group. rubin values (9-43 ± 4*3 versus 7-88 ± 4'8). These These data are of considerable importance as they results suggest that the unknown icterogenic factor refute the notion that hyperbilirubinaemia in was operating in a large enough percentage of the G-6-PD deficient neonates is always the result of population to shift the whole distribution curve of some noxious, albeit unidentified, exogenous haemo- neonatal hyperbilirubinaemia towards higher values. lytic agent. The combination of this factor with G-6-PD In populations with the Negro type of G-6-PD deficiency occurred with such frequency as to raise on September 27, 2021 by guest. Protected deficiency where the deficient infants do not have the incidence of severe neonatal jaundice in the higher bilirubin levels in comparison with the G-6-PD deficient infants to 43 %. normal controls (O'Flyn & Hsia, 1963; Zinkham, In another part of the world the existence of an 1963) severe neonatal jaundice develops if G-6-PD icterogenic factor was also demonstrated. Brown & deficiency is combined with prematurity (Capps et Wong (1965) showed differences in the peak serum al., 1963; Botha et al., 1967) or other contributory bilirubin values between British and Asiatic neonates factors (Levin, Charlton & Freiman, 1964; Hen- born in the same hospitals in Singapore. The peak drickse, 1965; Brown, 1966). serum bilirubin was 4-4 mg/100 ml for British, In a survey at Philadelphia (Oski, Eshagpour & 11.2±3-7 mg for Chinese, 10.0±3-3 mg for Malay Williams, 1966; Eshagpour, Oski & Williams, 1967) and 9-6 mg for the Indian newborns. In the same the combination in Negro infants of G-6-PD study it was also noticed that the peak serum bilirubin deficiency and prematurity produced levels of serum was reached later in Asiatic newborns. These observa- bilirubin above 20 mg/100 ml in seven out offourteen tions confirmed earlier reports that 'physiological Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
Neonatal jaundice 101 jaundice' was more frequent, more marked and of sonal material). (c) An unknown genetic factor longer duration among Chinese newborns (Lu, Lee acting in some families. (d) An unknown icterogenic & Chen, 1963; Wong, 1964). factor existing in some races or regions. Differences in the level of neonatal hyperbili- It is evident from the above that the explanation rubinaemia of full-term and even more of pre-term for the development of severe neonatal jaundice in infants existed between previous surveys (Hsia et some of the infants with G-6-PD deficiency should al., 1953; Dine, 1954; Obrinsky, Allen & Anderson, be sought outside the enzyme deficiency itself. 1954; Holman, 1958). Rightly they were ascribed Actually no difference was found in the enzyme either to poor standardization of laboratory methods characteristics between infants with G-6-PD de- for the estimation of bilirubin (Mather, 1960; ficiency who did and did not develop severe neo- Lucey, Phillips & McKay, 1960; Westphal, Viergiver natal jaundice (Kirkman et al., 1965). & Roth, 1962) and/or to factors in the medical and The unknown icterogenic factor existing in some nursing care of the newborns which could influence racial groups could operate either by increasing the bilirubin levels (Lucey, 1960). Such an explana- production or decreasing excretion of bilirubin (de- tion cannot be held for either the Greek or the layed induction of glucuronyl transferase activity or Singapore survey. Thus we have to accept that there inhibition of conjugation). This factor again could be are racial and regional differences in the level of either environmental in origin (food constituents, neonatal hyperbilirubinaemia. As for the mechanism trace elements, popular herbs, etc.) or genetic. The of these differences the evidence so far is incon- environmental origin seems unlikely as there clusive. In Lesbos, the group of infants with severe is very little in common between, for instance, jaundice but without incompatibility or G-6-PD Lesbos and Formosa, Greek and Chinese habits. If deficiency had as a whole lower haemoglobin values the factor is genetic it must be conferring a selective in comparison with the control group, yet, within the advantage in special environmental conditions to group, those that required an exchange transfusion balance out the disadvantage of the increased loss had significantly higher haemoglobin levels. Thus from kernicterus. It is difficult to see what possible we cannot be sure that haemolysis was the main selective advantage delayed maturation of the con- by copyright. mechanism for the development of the severe jugating capacity of the liver could have. The jaundice. Similarly the control group of the Lesbos possibility of a metabolic profile of the neonatal red survey (which had higher serum bilirubin values)had cells, that leads to a slight shortening of their life haemoglobin values both in the cord blood and on span, to have a selective advantage, is worth examin- the 4th day, higher than the corresponding Rhodes ing. survey group. Data on haemoglobin are not given The hypothesis proposed by Brewer & Powell for the surveys of Singapore and Formosa and thus (1965) is in this respect very attractive. These workers only hypotheses can be made for the mechanism of found an inverse relationship between erythrocyte increased 'physiological jaundice' in those popula- ATP levels and the time lapse for the development of tions. significant parasitaemia in experimental falciparum http://pmj.bmj.com/ malaria in non-immune subjects. It is likely that low levels of ATP lead to a shortening of the life span Conclusions and hypothesis of the erythrocyte so that the parasite does not The evidence so far mentioned can help us reach have enough time to complete its asexual cycle and a few conclusions as to the explanation for the vary- thus significant parasitaemia is delayed. There is ing risk of severe jaundice in G-6-PD deficient already evidence that ATP levels are under multi- newborns in different factorial control and on populations. depend the whole metabolic on September 27, 2021 by guest. Protected Glucose-6-phosphate dehydrogenase deficiency of profile of the red cells. the severe type produces a shortening of the life- Whatever the icterogenic factor may be it is very span of the red cells in the foetal and newborn likely from the data so far available (see Table 4) that period. This shortening is sufficient to lower the the G-6-PD deficiency gene and this factor are haemoglobin and raise the serum bilirubin values of mutually exclusive. A low incidence of unspecified the whole group but not enough to produce a sig- neonatal jaundice exists in the populations with high nificant reticulocyte response or frank anaemia. The frequency of G-6-PD deficiency, for instance non- increased rate of haemolysis of the group acquires Ashkenazi Jews, Negroes, Greeks of Rhodes, and clinical significance in a varying proportion of cases on the contrary in populations with increased neo- when combined with other icterogenic factors. A natal hyperbilirubinaemia the incidence of G-6-PD variety of such contributory factors have been identi- deficiency has been found to be low. Clearly there is fied so far: (a) Prematurity, affecting either the a need for more detailed information on the global excretion of bilirubin or the metabolism of the red epidemiology of neonatal jaundice and the problem cells or even both. (b) ABO incompatibility (per- is worthy of our attention from many aspects. Postgrad Med J: first published as 10.1136/pgmj.45.520.86 on 1 February 1969. Downloaded from
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104 Timos Valaes
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