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LOW ERYTHROCYTE PYRIDOXAL- KINASE ACTIVITY IN BLACKS: ITS POSSIBLE RELATION TO FALCIPARUM MALARIA
466
LOW ERYTHROCYTE PYRIDOXAL- KINASE ACTIVITY IN BLACKS: ITS POSSIBLE RELATION TO FALCIPARUM MALARIA S. K. MARTIN
L. H. MILLER
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, U.S.A.
J. A. KARK
C. U. HICKS M.
J.
HAUT
Department of Hematology, Walter Reed Army Institute of Research, Washington, DC, U.S.A. G.
V. C. OKOYE
J.
F. ESAN
Department of Hæmatology, University College Hospital, Ibadan, Nigeria The red-cell glucose-6-phosphate dehydrogenase (G.-6-P.D.) activity and red-cell pyridoxal-kinase (P.L.K.) activity of 27 Nigerian children with severe Plasmodium falciparum parasitæmia were compared with those of 26 healthy Nigerian children and 6 White adults. The mean P.L.K. activity of the malaria patients was similar to that of the Whites but significantly higher than that of the Nigerian controls. Correction for reduced mean red-cell age in patients was made by comparing the P.L.K.: G.-6-P.D. ratio for those subjects with stable G.-6-P.D. phenotypes. The mean P.L.K.: G.-6-P.D. ratio was the same for malaria patients and adult Whites but significantly higher than that for the Nigerian controls. These results suggest that the relatively high frequency of low red-cell P.L.K. activity among Blacks may have been selected for by falciparum malaria.
absorbed from the gut (pyridoxal, pyridoxamine, and pyridoxine).4 In the P.L.K. reaction, the terminal phosphoryl group of A.T.P. is exchanged with the 5’-hydroxyl group of the forms of B6 vitamins absorbed from the gut.4 Compared to other racial groups whose genetic characteristics have not been influenced by malaria, Blacks have a high frequency of the low-activity P.L.K. variant in red cells.3,5,6 This genetic variant appears to be inherited as a single autosomal allele,3.6 and confers decreased in-vivo stability on the red-cell P.L.K.3 We have studied an African population in Ibadan, Nigeria, whose genetic characteristics have been influenced by holoendemic falciparum malaria. We have compared the levels of red-cell P.L.K. activity of children with severe falciparum parasitaemia with those of uninfected children in the same population in order to determine whether low red-cell P.L.K. activity is less common in children experiencing life-threatening falciparum in-
fections.
Summary
Introduction DEATH from falciparum malaria is closely related to the level of parasitaemia, which usually reaches 100 000 parasites/1 of blood in fatal cases.’ Any genetic traits which suppress parasitsemia (e.g., sickle-cell trait) would increase chances of survival.2 Hence, such genes would increase in frequency in populations among which there is holoendemic malaria.2 Low red-cell pyridoxal-kinase (P.L.K.) activity may be such a trait.3 P.L.K. catalyses the major step in the synthesis of B6 coenzymes (pyridoxal 5’-phosphate and pyridoxamine 5’-phosphate) from those forms of vitamin B6 which are
11. 12. 13.
Ling, C. M., Overby, L. R. J. Immun. 1972, 109, 834. Hollinger, F., Vorndam, V., Dreesman, G. ibid. 1971, 107, 1099. Hoofnagle, J. H., Gerety, R. J., Barker, L. F. in Transmissible Disease and Blood Transfusion (edited by T. J. Greenwalt and G. A. Jamieson); p. 43.
New York, 1975. 14. Hootnagle, J. H., Gerety, R. J., Barker, L. F. Lancet, 1973, ii, 869. 15. Magnius, L. O. Clin. exp. Immun. 1975, 20, 209. 16. Tabor, E., Gerety, R. J., Barker, L. F. J infect. Dis. 1977, 136, 541. 17. Moritsugu, Y., Dienstag, J. L., Valdesuso, J., Wong, D. C., Wagner, J., Routenberg, J. A., Purcell, R. H. Infect. Immun. 1976, 13, 898. 18. Miller, W. J., Provost, P. J., McAleer, W. J., Ittensohn, O. L., Villarejos, V. M., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1975, 149, 254. 19. Chiang, W. B., Wentworth, B. B., Alexander, E. R. J. Immun. 1970, 104, 992. 20. Smith, K. O., Gehle, W. D., McCracken, A. W. J. Immunol. Methods, 1974,
5, 337. 21.
Henle, G., Henle, W. J. Bact. 1966, 91, 1248.
Patients and Methods Patients and Controls From children
5
-
Sept. 23, 1976, the blood films of Nigerian the outpatient department of University College Hospital, Ibadan, with rectal temperatures above 38.5OC were screened for falciparum parasites. 65 children with parasitaemia of 1% and above were accepted into the study, 61 of whom had an acute illness which started 4 days or less before presentation. They were treated with aspirin and chloroquine for two to three days, and then with weekly pyrimethamine for four weeks. The red-cell glucose-6phosphate dehydrogenase (G.-6-P.D.) activity, G.-6-P.D. electrophoretic pattern, and red-cell P.L.K. activity were determined before or during the first four days of treatment for 9 patients (the acute patient group). To exclude the possibility that alterations in red-cell P.L.K. activity were due to the acute infection, P.L.K. activity was measured four to eight weeks after initiation of treatment in 18 patients who returned for follow-up (the convalescent group). The mean hsematocrit readings before treatment were 24·8+_2·4 (S.E.M.) for the acute group and
July
seen
to
at
mean haematocrit were convalescent children for the 33-5±0-9. The readings mean levels of parasitaemia in the acute and convalescent patients were (2-16±0-46)xl0’’ and (1.49±0.26)x105 parasitised red cells/.1 blood respectively; levels above 100000/ were seen in 7 and 12 patients in the acute and convalescent groups respectively. The controls were 26 healthy, at’ebrile, Nigerian children attending local immunisation and well-baby clinics in Ibadan. The control children had no malaria parasites in their blood films, and belonged to the same age-range and socioeconomic groups as the patients and had mean haematocrit readings of 32.0±2.2% (s.E.M.). 6 healthy Whites on weekly pyrimethamine for malaria prophylaxis acted as additional controls for P.L.K. Repeated red-cell P.L.K. measurements were made on blood from 2 of our team before and after initiation of weekly chloroquine prophylaxis and no change in P.L.K. activity was detected.
27-4±1-2for the convalescent group. The
Methods Red-cell P.L.K. activity was measured by a modification of the method of Lumeng and Li.7 Instead of measuring the P.L.K.-reaction product, pyridoxal phosphate, by the tyrosineapodecarboxylase method, we used a fluorometric method in which pyridoxal phosphate was oxidised to 4-pyridoxic-acid 5’-phosphate,8 which was then measured fluorometrically.9,IO Measurements of normal red-cell P.L.K. activity by both the
467 and the fluorometric assay for pyriresults."I identical doxal phosphate give In case P.L.K. activity in the patient population may have been influenced by the younger mean red-cell age of the convalescent children’ G.-6-P.D. activity was measuredl2 as a marker for red-cell age. The two prevalent normal-activity G.-6-P.D. variants in the area (GdB, Gd^) were identified by electrophoresis." Any subject with G.-6-P.D. activity below 7-5 I.u./g Hb was considered to have the deficient variant, GdA-. Any patient with red-cell G.-6-P.D. above two standard deviations of the mean for the control group (>20 i.u./g Hb) was excluded from the data analysis. Further correction for variation in redcell age was made by examining the ratio of red-cell P.L.K. to G.-6-P.D., two red-cell age-dependent enzymes. Ratios were not calculated for the G.-6-P.D.-deficient individuals.
apodecarboxylase method
Results
Red-cell P.L.K. was plotted as a function of red-cell G.-6-P.D. for malaria patients and Nigerian controls (fig. 1). Only 3 malaria patients (1 acute and 2 convalescent) had G.-6-P.D. levels above two standard deviations of the mean for the control group, and they were excluded from the analysis. At any G.-6-P.D. level, control children in general had lower red-cell P.L.K. than the patients (fig. 1). There was no significant increase in mean P.L.K. with increasing G.-6-P.D. for either patients or controls. By Spearman rank test there was no correlation between P.L.K. and G.-6-P.D. for either patient (P=0-44) or control (P=045) groups. The mean red-cell G.-6-P.D. (+S.E.M.) of the 16 convalescent patients was 2.12±0-21 mi.u./g Hb and 2.10+0.22 mI.u./g Hb for the 8 acute patients. The mean red-cell P.L.K. of the 6-White controls (194+009 mi.u./g Hb) was not significantly different from the means of patient groups. By contrast, the 26
Fig. 2-Ratio of red-cell pyridoxal-kinase activity to glucose-6phosphate dehydrogenase activity of malaria patients and controls, showing their mean:t S.E.M.
Nigerian controls had a significantly lower mean red-cell P.L.K. (1-32+0.10 mi.u./g Hb) than the patients (P<0.01) and the White controls (P<0.01) measured by Student’s t test. The ratio of red-cell P.L.K. to G.-6-P.D. was examined for those individuals with normal G.-6-P.D. activity (fig. 2). This ratio was similar for the 12 convalescent patients (mean±S.E.M., 0162±0023x10-3), the 4 acute patients (0196±0048x10-3), and the 6 White controls (0.178±0.009×10-3). However, the mean ratio for the Nigerian controls (0.098±0.008×10-3) was significantly lower (P<0.01). Discussion
Fig. 1-Relation between red-cell pyridoxal-kinase activity and gtucose-6-phosphate dehydrogenase activity for three groups of Nigerian children.
The pyridoxal-kinase activity of the malaria patients with severe parasitaemia was significantly higher than that of the Nigerian controls of similar age and socioeconomic background. The difference in enzyme-activity between the 2 groups may have been genetically determined, suggesting an increased susceptibility to severe falciparum malaria in children with high erythrocyte enzyme activity.14 Alternatively, the higher P.L.K. activity in the malaria patients could have been the effect of the acute infection due either to a change in the age of circulating red cells" or to direct effects of the parasite on red-cell metabolism; in the convalescent period, there would be a relatively high proportion of young red-cells because of recovery from haemolysis. We have attempted to correct for any difference in red-cell age distribution by calculating the ratio of P.L.K. to G.-6-P.D., both P.L.K. and G.-6-P.D. being red-cell age-dependent enzymes. Proof that the higher enzyme activity in malaria patients is a genetic trait will require long-term follow-up of the patient-population or the use of another marker for a deficient enzyme that is independent of its activity--e.g., a difference in electrophoretic mobility. However, at present the low-activity P.L.K. variant is indistinguishable from the normal enzyme except for its lower activity in older red cells.3 Factors such as diet or effect of antimalarial drugs do not seem to account for the difference. The malaria patients and Nigerian controls were from the same socioeconomic group, with no discernible differences in diet. The two antimalarial drugs used in the study, chloro-
468
pyrimethamine, do not seem to increase pyridoxal-kinase activity. Weekly chloroquine did not inqume and
the enzyme levels in the 2 members of our team from the United States. All 6 Whites studied were on weekly pyrimethamine and had comparable enzyme levels to Whites in the United States. Also, acute and convalescent patients had comparable red-cell P.L.K. activities. If the higher erythrocyte pyridoxal-kinase activity predisposed to severe falciparum malaria, it may be because the parasite lacks this enzyme and is dependent on the erythrocyte enzyme for its pyridoxal-kinase requirements. Perhaps pyridoxal phosphate may stimulate extraerythrocytic growth of P. falciparum in the same way that coenzyme A enhanced the extra-erythrocytic growth of P. lophoræ.16,17 Continuous culture of P. falciparum in human erythrocytes in vitro has recently been accomplished.18,19 Knowledge about the extra-erythrocytic requirements of the parasite may provide insight into methods for extracellular cultivation. crease
This study was partly supported by the World Health Organisation. We thank Prof. B. 0. Osunkoya, Dr G. I. Adeleye, Dr F. A. Adekunle,’ and Mr C. Ugbode of the University College Hospital, Ibadan ; Dr Mary Deane and staff of St. Mary’s Hospital, Eleta, Ibadan; and Prof. E. 0. Ogunlana of the faculty of pharmacy, University of Ife, Ife, Nigeria. We also thank Dr David Alling for help with the statistical
analysis. Requests for reprints to S. K. M.
way into the scrotum were merely retractile was questioned. Such testes were deviated by inguinal tethering. The spermatic cord was short enough in over half the tethered testes to require retroperitoneal dissection to achieve the fully descended position, not a significantly different proportion compared with the some
obstructed group. Introduction DISCUSSION of testicular position is complicated by in definition; intermediate types confuse diagnosis and occasionally even the physical signs seem inconstant. In general, testes away from the low scrotal position may be arrested along the normal line of descent, deviated from that normal line, or retractile. "Deviated" is preferrable to "ectopic" which implies any form of displacement, and "arrested" avoids the imprecision of "undescended". Operation in the first five years of life is widely accepted for arrested descent, for the rarer deviated types, and for superficial inguinal-pouch testes which cannot be pushed into the scrotum at all (deviated/obstructed). On the other hand, testes in the low scrotal position which retract readily to the external ring do not
difficulty
require operation. More debatable are testes which lie persistently lateral the external ring, and which can be coaxed passively some way into the scrotum but will not stay there. Browne,’ and Scorer and Farrington,2 who say that these superficial inguinal-pouch testes are merely retractile if they can be pushed into the scrotum, imply that since descent is not obstructed (by scrotal neck fibrosis), operation is unnecessary, though Scorer3 has noted that many "retractile" testes fail to reach the adult position to
REFERENCES
1. Field, J. W. Trans. R. Soc. trop. Med. Hyg. 1949, 43, 33. 2. Allison, A. C. Ann. N.Y. Acad. Sci. 1961, 91, 710. 3. Chern, C. J., Beutler, E. Am. J. hum. Gen. 1976, 28, 9. 4. Snell, E. E., Haskell, B. E. in Comprehensive Biochemistry (edited by M. Florkin and E. H. Stotzl); p. 47, Amsterdam. 1971. 5. Chern, C. J., Beutler, E. Science, 1975, 187, 1084. 6. Anderson, B. B., Mollin, D. L., Child, J. A., Modell, C. B., Perry, G. M. in Iron Metabolism and Its Disorders (edited by H. Kief); p. 241. Amster-
dam, 1975. 7. 8. 9. 10. 11.
Lumeng, L., Li, T. K. J. clin. Invest. 1974, 53, 693. Ohishi, N., Fukui, S. Archs Biochem. Biophys. 1968, 128, 606. Bonavita, V. ibid. 1960, 88, 366. Adams, E. Analyt. Biochem. 1969, 31, 118. Kark, J. A., Haut, M. J., Hicks, C. U., McQuilkin, C. T. Unpublished. 12. Tech. Rep. Ser. Wld Hlth Org. 1967, no. 366. 13. Luzzatto, L., Afolayan, A. J. clin. Invest. 1968, 47, 1833. 14. Bienzle, U., Ayeni, O., Lucas, A. O., Luzzato, L. Lancet, 1972, i, 107. 15. Wilson, R. J. M. Wld Hlth Org. (in the press). 16. Trager, W. J. exp. Med. 1952, 96, 456. 17. Trager, W., J. Protozool. 1954, i, 231. 18. Trager, W., Jensen, J. B., Science, 1976, 193, 673. 19. Haynes, J. D. et al. Nature, 1976, 263, 767.
by puberty. However, Smith,4 doctor
to Rugby School, could not how cremasteric retraction could rotate a testis by 180°and cause it to lie out in the superficial inguinal pouch. Experience shows that in boys with testes in normal positions, the cremasteric reflex does not cause the testis to move lateral to the external ring. An alternative explanation for the lateral deviation is that adhesions between the cord coverings and the inguinal region hitch the testis laterally when the cremaster contracts. An analogue of this deviated, tethered group is a loop of see
(cremaster) passing through an opening (external and held to one side just beyond the ring (by lateral adhesions). When the loop is long, the movable pulley (testis) round which it passes dangles, downshortens the rope and the testis swings laterally. These lateral adhesions are easily shown at operation on such testes. The concept of tethering, as described by Lockwood5 and by Backhouse,6 also explains the rarer types of deviated testis ("ectopia"). rope
ring)
DIAGNOSIS AND MANAGEMENT OF TESTES IN THE SUPERFICIAL INGUINAL POUCH E. C. ASHBY St. Richard’s
Hospital, Chichester,
West Sussex PO19 4SE
A physical sign was assessed and operation findings were noted in boys with testes which tended to deviate lateral to the external ring who were seen over a five-year period. Tensing the abdominal muscles by straight-leg raising not only facilitated palpation of such testes but also helped to demonstrate lateral deviation. The view that testes found lateral to the external ring and which could be pushed
Summary
One source of confusion in the literature is the lack of a clear distinction between testes at the external ring and those lateral to it. For example, Browne defined the superficial inguinal pouch as the space between the external oblique and Scarpa’s fascia, but two paragraphs later described the "emergent" testis (which lies at or just below the external ring) as being within the pouch. Any classification of abnormally placed testes is bound to be oversimplified because of hybrid or intermediate forms. For practical purposes there is much to be
LOW ERYTHROCYTE PYRIDOXAL- KINASE ACTIVITY IN BLACKS: ITS POSSIBLE RELATION TO FALCIPARUM MALARIA S. K. MARTIN
L. H. MILLER
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, U.S.A.
J. A. KARK
C. U. HICKS M.
J.
HAUT
Department of Hematology, Walter Reed Army Institute of Research, Washington, DC, U.S.A. G.
V. C. OKOYE
J.
F. ESAN
Department of Hæmatology, University College Hospital, Ibadan, Nigeria The red-cell glucose-6-phosphate dehydrogenase (G.-6-P.D.) activity and red-cell pyridoxal-kinase (P.L.K.) activity of 27 Nigerian children with severe Plasmodium falciparum parasitæmia were compared with those of 26 healthy Nigerian children and 6 White adults. The mean P.L.K. activity of the malaria patients was similar to that of the Whites but significantly higher than that of the Nigerian controls. Correction for reduced mean red-cell age in patients was made by comparing the P.L.K.: G.-6-P.D. ratio for those subjects with stable G.-6-P.D. phenotypes. The mean P.L.K.: G.-6-P.D. ratio was the same for malaria patients and adult Whites but significantly higher than that for the Nigerian controls. These results suggest that the relatively high frequency of low red-cell P.L.K. activity among Blacks may have been selected for by falciparum malaria.
absorbed from the gut (pyridoxal, pyridoxamine, and pyridoxine).4 In the P.L.K. reaction, the terminal phosphoryl group of A.T.P. is exchanged with the 5’-hydroxyl group of the forms of B6 vitamins absorbed from the gut.4 Compared to other racial groups whose genetic characteristics have not been influenced by malaria, Blacks have a high frequency of the low-activity P.L.K. variant in red cells.3,5,6 This genetic variant appears to be inherited as a single autosomal allele,3.6 and confers decreased in-vivo stability on the red-cell P.L.K.3 We have studied an African population in Ibadan, Nigeria, whose genetic characteristics have been influenced by holoendemic falciparum malaria. We have compared the levels of red-cell P.L.K. activity of children with severe falciparum parasitaemia with those of uninfected children in the same population in order to determine whether low red-cell P.L.K. activity is less common in children experiencing life-threatening falciparum in-
fections.
Summary
Introduction DEATH from falciparum malaria is closely related to the level of parasitaemia, which usually reaches 100 000 parasites/1 of blood in fatal cases.’ Any genetic traits which suppress parasitsemia (e.g., sickle-cell trait) would increase chances of survival.2 Hence, such genes would increase in frequency in populations among which there is holoendemic malaria.2 Low red-cell pyridoxal-kinase (P.L.K.) activity may be such a trait.3 P.L.K. catalyses the major step in the synthesis of B6 coenzymes (pyridoxal 5’-phosphate and pyridoxamine 5’-phosphate) from those forms of vitamin B6 which are
11. 12. 13.
Ling, C. M., Overby, L. R. J. Immun. 1972, 109, 834. Hollinger, F., Vorndam, V., Dreesman, G. ibid. 1971, 107, 1099. Hoofnagle, J. H., Gerety, R. J., Barker, L. F. in Transmissible Disease and Blood Transfusion (edited by T. J. Greenwalt and G. A. Jamieson); p. 43.
New York, 1975. 14. Hootnagle, J. H., Gerety, R. J., Barker, L. F. Lancet, 1973, ii, 869. 15. Magnius, L. O. Clin. exp. Immun. 1975, 20, 209. 16. Tabor, E., Gerety, R. J., Barker, L. F. J infect. Dis. 1977, 136, 541. 17. Moritsugu, Y., Dienstag, J. L., Valdesuso, J., Wong, D. C., Wagner, J., Routenberg, J. A., Purcell, R. H. Infect. Immun. 1976, 13, 898. 18. Miller, W. J., Provost, P. J., McAleer, W. J., Ittensohn, O. L., Villarejos, V. M., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1975, 149, 254. 19. Chiang, W. B., Wentworth, B. B., Alexander, E. R. J. Immun. 1970, 104, 992. 20. Smith, K. O., Gehle, W. D., McCracken, A. W. J. Immunol. Methods, 1974,
5, 337. 21.
Henle, G., Henle, W. J. Bact. 1966, 91, 1248.
Patients and Methods Patients and Controls From children
5
-
Sept. 23, 1976, the blood films of Nigerian the outpatient department of University College Hospital, Ibadan, with rectal temperatures above 38.5OC were screened for falciparum parasites. 65 children with parasitaemia of 1% and above were accepted into the study, 61 of whom had an acute illness which started 4 days or less before presentation. They were treated with aspirin and chloroquine for two to three days, and then with weekly pyrimethamine for four weeks. The red-cell glucose-6phosphate dehydrogenase (G.-6-P.D.) activity, G.-6-P.D. electrophoretic pattern, and red-cell P.L.K. activity were determined before or during the first four days of treatment for 9 patients (the acute patient group). To exclude the possibility that alterations in red-cell P.L.K. activity were due to the acute infection, P.L.K. activity was measured four to eight weeks after initiation of treatment in 18 patients who returned for follow-up (the convalescent group). The mean hsematocrit readings before treatment were 24·8+_2·4 (S.E.M.) for the acute group and
July
seen
to
at
mean haematocrit were convalescent children for the 33-5±0-9. The readings mean levels of parasitaemia in the acute and convalescent patients were (2-16±0-46)xl0’’ and (1.49±0.26)x105 parasitised red cells/.1 blood respectively; levels above 100000/ were seen in 7 and 12 patients in the acute and convalescent groups respectively. The controls were 26 healthy, at’ebrile, Nigerian children attending local immunisation and well-baby clinics in Ibadan. The control children had no malaria parasites in their blood films, and belonged to the same age-range and socioeconomic groups as the patients and had mean haematocrit readings of 32.0±2.2% (s.E.M.). 6 healthy Whites on weekly pyrimethamine for malaria prophylaxis acted as additional controls for P.L.K. Repeated red-cell P.L.K. measurements were made on blood from 2 of our team before and after initiation of weekly chloroquine prophylaxis and no change in P.L.K. activity was detected.
27-4±1-2for the convalescent group. The
Methods Red-cell P.L.K. activity was measured by a modification of the method of Lumeng and Li.7 Instead of measuring the P.L.K.-reaction product, pyridoxal phosphate, by the tyrosineapodecarboxylase method, we used a fluorometric method in which pyridoxal phosphate was oxidised to 4-pyridoxic-acid 5’-phosphate,8 which was then measured fluorometrically.9,IO Measurements of normal red-cell P.L.K. activity by both the
467 and the fluorometric assay for pyriresults."I identical doxal phosphate give In case P.L.K. activity in the patient population may have been influenced by the younger mean red-cell age of the convalescent children’ G.-6-P.D. activity was measuredl2 as a marker for red-cell age. The two prevalent normal-activity G.-6-P.D. variants in the area (GdB, Gd^) were identified by electrophoresis." Any subject with G.-6-P.D. activity below 7-5 I.u./g Hb was considered to have the deficient variant, GdA-. Any patient with red-cell G.-6-P.D. above two standard deviations of the mean for the control group (>20 i.u./g Hb) was excluded from the data analysis. Further correction for variation in redcell age was made by examining the ratio of red-cell P.L.K. to G.-6-P.D., two red-cell age-dependent enzymes. Ratios were not calculated for the G.-6-P.D.-deficient individuals.
apodecarboxylase method
Results
Red-cell P.L.K. was plotted as a function of red-cell G.-6-P.D. for malaria patients and Nigerian controls (fig. 1). Only 3 malaria patients (1 acute and 2 convalescent) had G.-6-P.D. levels above two standard deviations of the mean for the control group, and they were excluded from the analysis. At any G.-6-P.D. level, control children in general had lower red-cell P.L.K. than the patients (fig. 1). There was no significant increase in mean P.L.K. with increasing G.-6-P.D. for either patients or controls. By Spearman rank test there was no correlation between P.L.K. and G.-6-P.D. for either patient (P=0-44) or control (P=045) groups. The mean red-cell G.-6-P.D. (+S.E.M.) of the 16 convalescent patients was 2.12±0-21 mi.u./g Hb and 2.10+0.22 mI.u./g Hb for the 8 acute patients. The mean red-cell P.L.K. of the 6-White controls (194+009 mi.u./g Hb) was not significantly different from the means of patient groups. By contrast, the 26
Fig. 2-Ratio of red-cell pyridoxal-kinase activity to glucose-6phosphate dehydrogenase activity of malaria patients and controls, showing their mean:t S.E.M.
Nigerian controls had a significantly lower mean red-cell P.L.K. (1-32+0.10 mi.u./g Hb) than the patients (P<0.01) and the White controls (P<0.01) measured by Student’s t test. The ratio of red-cell P.L.K. to G.-6-P.D. was examined for those individuals with normal G.-6-P.D. activity (fig. 2). This ratio was similar for the 12 convalescent patients (mean±S.E.M., 0162±0023x10-3), the 4 acute patients (0196±0048x10-3), and the 6 White controls (0.178±0.009×10-3). However, the mean ratio for the Nigerian controls (0.098±0.008×10-3) was significantly lower (P<0.01). Discussion
Fig. 1-Relation between red-cell pyridoxal-kinase activity and gtucose-6-phosphate dehydrogenase activity for three groups of Nigerian children.
The pyridoxal-kinase activity of the malaria patients with severe parasitaemia was significantly higher than that of the Nigerian controls of similar age and socioeconomic background. The difference in enzyme-activity between the 2 groups may have been genetically determined, suggesting an increased susceptibility to severe falciparum malaria in children with high erythrocyte enzyme activity.14 Alternatively, the higher P.L.K. activity in the malaria patients could have been the effect of the acute infection due either to a change in the age of circulating red cells" or to direct effects of the parasite on red-cell metabolism; in the convalescent period, there would be a relatively high proportion of young red-cells because of recovery from haemolysis. We have attempted to correct for any difference in red-cell age distribution by calculating the ratio of P.L.K. to G.-6-P.D., both P.L.K. and G.-6-P.D. being red-cell age-dependent enzymes. Proof that the higher enzyme activity in malaria patients is a genetic trait will require long-term follow-up of the patient-population or the use of another marker for a deficient enzyme that is independent of its activity--e.g., a difference in electrophoretic mobility. However, at present the low-activity P.L.K. variant is indistinguishable from the normal enzyme except for its lower activity in older red cells.3 Factors such as diet or effect of antimalarial drugs do not seem to account for the difference. The malaria patients and Nigerian controls were from the same socioeconomic group, with no discernible differences in diet. The two antimalarial drugs used in the study, chloro-
468
pyrimethamine, do not seem to increase pyridoxal-kinase activity. Weekly chloroquine did not inqume and
the enzyme levels in the 2 members of our team from the United States. All 6 Whites studied were on weekly pyrimethamine and had comparable enzyme levels to Whites in the United States. Also, acute and convalescent patients had comparable red-cell P.L.K. activities. If the higher erythrocyte pyridoxal-kinase activity predisposed to severe falciparum malaria, it may be because the parasite lacks this enzyme and is dependent on the erythrocyte enzyme for its pyridoxal-kinase requirements. Perhaps pyridoxal phosphate may stimulate extraerythrocytic growth of P. falciparum in the same way that coenzyme A enhanced the extra-erythrocytic growth of P. lophoræ.16,17 Continuous culture of P. falciparum in human erythrocytes in vitro has recently been accomplished.18,19 Knowledge about the extra-erythrocytic requirements of the parasite may provide insight into methods for extracellular cultivation. crease
This study was partly supported by the World Health Organisation. We thank Prof. B. 0. Osunkoya, Dr G. I. Adeleye, Dr F. A. Adekunle,’ and Mr C. Ugbode of the University College Hospital, Ibadan ; Dr Mary Deane and staff of St. Mary’s Hospital, Eleta, Ibadan; and Prof. E. 0. Ogunlana of the faculty of pharmacy, University of Ife, Ife, Nigeria. We also thank Dr David Alling for help with the statistical
analysis. Requests for reprints to S. K. M.
way into the scrotum were merely retractile was questioned. Such testes were deviated by inguinal tethering. The spermatic cord was short enough in over half the tethered testes to require retroperitoneal dissection to achieve the fully descended position, not a significantly different proportion compared with the some
obstructed group. Introduction DISCUSSION of testicular position is complicated by in definition; intermediate types confuse diagnosis and occasionally even the physical signs seem inconstant. In general, testes away from the low scrotal position may be arrested along the normal line of descent, deviated from that normal line, or retractile. "Deviated" is preferrable to "ectopic" which implies any form of displacement, and "arrested" avoids the imprecision of "undescended". Operation in the first five years of life is widely accepted for arrested descent, for the rarer deviated types, and for superficial inguinal-pouch testes which cannot be pushed into the scrotum at all (deviated/obstructed). On the other hand, testes in the low scrotal position which retract readily to the external ring do not
difficulty
require operation. More debatable are testes which lie persistently lateral the external ring, and which can be coaxed passively some way into the scrotum but will not stay there. Browne,’ and Scorer and Farrington,2 who say that these superficial inguinal-pouch testes are merely retractile if they can be pushed into the scrotum, imply that since descent is not obstructed (by scrotal neck fibrosis), operation is unnecessary, though Scorer3 has noted that many "retractile" testes fail to reach the adult position to
REFERENCES
1. Field, J. W. Trans. R. Soc. trop. Med. Hyg. 1949, 43, 33. 2. Allison, A. C. Ann. N.Y. Acad. Sci. 1961, 91, 710. 3. Chern, C. J., Beutler, E. Am. J. hum. Gen. 1976, 28, 9. 4. Snell, E. E., Haskell, B. E. in Comprehensive Biochemistry (edited by M. Florkin and E. H. Stotzl); p. 47, Amsterdam. 1971. 5. Chern, C. J., Beutler, E. Science, 1975, 187, 1084. 6. Anderson, B. B., Mollin, D. L., Child, J. A., Modell, C. B., Perry, G. M. in Iron Metabolism and Its Disorders (edited by H. Kief); p. 241. Amster-
dam, 1975. 7. 8. 9. 10. 11.
Lumeng, L., Li, T. K. J. clin. Invest. 1974, 53, 693. Ohishi, N., Fukui, S. Archs Biochem. Biophys. 1968, 128, 606. Bonavita, V. ibid. 1960, 88, 366. Adams, E. Analyt. Biochem. 1969, 31, 118. Kark, J. A., Haut, M. J., Hicks, C. U., McQuilkin, C. T. Unpublished. 12. Tech. Rep. Ser. Wld Hlth Org. 1967, no. 366. 13. Luzzatto, L., Afolayan, A. J. clin. Invest. 1968, 47, 1833. 14. Bienzle, U., Ayeni, O., Lucas, A. O., Luzzato, L. Lancet, 1972, i, 107. 15. Wilson, R. J. M. Wld Hlth Org. (in the press). 16. Trager, W. J. exp. Med. 1952, 96, 456. 17. Trager, W., J. Protozool. 1954, i, 231. 18. Trager, W., Jensen, J. B., Science, 1976, 193, 673. 19. Haynes, J. D. et al. Nature, 1976, 263, 767.
by puberty. However, Smith,4 doctor
to Rugby School, could not how cremasteric retraction could rotate a testis by 180°and cause it to lie out in the superficial inguinal pouch. Experience shows that in boys with testes in normal positions, the cremasteric reflex does not cause the testis to move lateral to the external ring. An alternative explanation for the lateral deviation is that adhesions between the cord coverings and the inguinal region hitch the testis laterally when the cremaster contracts. An analogue of this deviated, tethered group is a loop of see
(cremaster) passing through an opening (external and held to one side just beyond the ring (by lateral adhesions). When the loop is long, the movable pulley (testis) round which it passes dangles, downshortens the rope and the testis swings laterally. These lateral adhesions are easily shown at operation on such testes. The concept of tethering, as described by Lockwood5 and by Backhouse,6 also explains the rarer types of deviated testis ("ectopia"). rope
ring)
DIAGNOSIS AND MANAGEMENT OF TESTES IN THE SUPERFICIAL INGUINAL POUCH E. C. ASHBY St. Richard’s
Hospital, Chichester,
West Sussex PO19 4SE
A physical sign was assessed and operation findings were noted in boys with testes which tended to deviate lateral to the external ring who were seen over a five-year period. Tensing the abdominal muscles by straight-leg raising not only facilitated palpation of such testes but also helped to demonstrate lateral deviation. The view that testes found lateral to the external ring and which could be pushed
Summary
One source of confusion in the literature is the lack of a clear distinction between testes at the external ring and those lateral to it. For example, Browne defined the superficial inguinal pouch as the space between the external oblique and Scarpa’s fascia, but two paragraphs later described the "emergent" testis (which lies at or just below the external ring) as being within the pouch. Any classification of abnormally placed testes is bound to be oversimplified because of hybrid or intermediate forms. For practical purposes there is much to be