709 min-K deficiency of which no traces persist after 85 h would result in a serious drop of coagulation factors because: (a) vitamin-K deficiency develops slowly, so inhibition of coagulation factor synthesis will set in gradually, and (b) coagulation factor activity does not immediately respond to an inhibitor of coagulation factor synthesis, but lags behind because the factors already circulating take some time to disappear. In the rat, which has a high turnover rate of coagulation factors8 it still takes 7 days on a vitamin-K-deficient diet before a bleeding tendency develops.9 In normal man complete inhibition that leaves no trace of clotting-factor synthesis after 85 h can cause a fall in prothrombin of more than 20%.’° We suggest that the blood of a newborn with a bleeding tendency be analysed for vitamin-K deficiency (staphylocoagulase-determined factor ii much greater than one-stage estimation), and heparin-like inhibitor (protamine-sulphate-induced reduction of functional antithrombin 3), and that therapeutic measures are taken accordingly. We would be happy to analyse any samples that suggest presence of vitamin-K deficiency in the newborn.
indirect immunofluorescence and anticomplement immunofluorescence for the presence of T antibody. All the sera were reacted against a battery of cell lines that included the NMK line nude-mouse kidney cells transformed by BK virus, the VA-2 line (human fibroblast transformed by SV40), and the WI-38 line (diploid human fibroblasts). Of 113 sera tested (see table) 6 had non-specific antinuclear antibodies and 12 gave weak reactions with the viral-transformed cells at a 1/2 dilution. The 12 weakly positive sera were further tested by anticomplement methods at higher dilutions. None of these 12 sera was positive by the anticomplement method. The low titres of reactivity and the lack of complement fixation argue against a specific antigen-antibody reaction at the low dilution in the initial screening. Corallini et al .2 have reported that 11 patients with tumours out of 952 examined had antibodies to BK virus T antigen. Had we examined more sera we also might have found some cancer patients with positive reactions. Nevertheless, although the possibility that human papovaviruses may be associated with a small number of tumours cannot be ruled out, our data suggest a lack of widespread association of these viruses with human neoplasia.
Gusthuis, Amsterdam, Netherlands
Laboratory of Pathology, Division of Cancer Biology and Diagnosis,
Onze Lieve Vrouwen
of Biochemistry,
Department Rijksuniversiteit Limburg
J. M.
VAN
DOORM
H. C. HEMKER
National Cancer Institute,
Bethesda, Maryland 20014, U.S.A.
J. COSTA C. YEE A. S. RABSON
POLYOMA VIRUS IN URINE DURING PREGNANCY
ABSENCE OF PAPOVAVIRUS T ANTIBODY IN PATIENTS WITH MALIGNANCIES
SiR,—JC virus and BK virus, the two prototype viruses of the human polyoma group, are ubiquitous.’ They infect large numbers of people, and in most cases seroconversion takes place in childhood. The ability of these viruses to transform cells in vitro and to produce tumours in laboratory animals has led to suggestions that they may play a role in the aetiology of human neoplasms. ANTIBODIES TO VIRAL T ANTIGENS IN PATIENTS WITH TUMOURS OR BENIGN CONDITIONS AND CONTROLS
Animals bearing tumours induced by BK or JC virus have antibodies to a viral antigen present in the nucleus of the tumour cell. These "T" antigens and antibodies can be detected by immunofluorescence and by complement fixation; BK and JC virus T antigens high degree of cross-reactivity with each other and with the T antigen ofSV40 virus. If any human tumours are caused by BK or JC virus some patients should have antibody to the T antigens of these viruses in their sera. We have tested a panel of human sera by 8. Suttie, J. W. Science, 1973, 179, 192. 9.van Oosterom, A. T., Mattie, H., Hermens, W. Th., Veltkamp, J. J. Thrombos. Hœm. 1976,35,607. 10 van der Meer, J., Hemker, H. C., Loeliger, E. A. Thrombos. Diathes. hœmorrh. 1968, suppl. 19, 1. 1.Padgett, B.L., Walker, D. L. Progr. med. Virol. 1976, 22, 1.
SIR,-Active infection with the human polyomaviruses BK and JC has been recorded only in patients whose immunological responses have been impaired. Our attempts to isolate these viruses from the urinary tract, throat washings, or faeces of children under the age of 15 years who seem immunologically competent have been unsuccessful, despite the fact that antibody studies have shown that most people have been exposed to these viruses and that primary infection occurs most frequently in childhood. In November, 1976, we started a prospective study of 1000 patients at St Mary’s Hospital. We chose pregnant women because of their apparent susceptibility to virus infection, perhaps due to physiological changes in pregnancy. We isolated human polyomavirus JC from the urinary tract of a 38-year-old woman who came to the antenatal clinic on Dec. 12, 1976, in the 36th week of her third pregnancy. Her obstetric history included a spontaneous abortion at 7 weeks in 1972 and the delivery of a normal full-term female infant in 1974. Her third pregnancy proceeded uneventfully to 41 weeks when a normal female infant weighing 3200 g was delivered by spontaneous vertex delivery. The placenta weighed 520 g and appeared normal, as did the cord and membranes. Jaundice developed in the baby after 36 h, and the serum-bilirubin was 176 fLmol/1 rising to 220 fLmol/1 on the sixth day. No evidence of rhesus incompatibility or other cause for the jaundice was found, and the jaundice settled after phototherapy. Maternal infection with a human polyomavirus was suspected as a result of routine light microscopy of stained smears of urinary sediment. A few atypical urothelial cells with large basophilic intranuclear inclusions were observed in cytological smears prepared from urine samples collected at 36, 38, 39, 40, and 41 weeks of pregnancy. A small number of papovavirus particles were observed under the electron microscope in negatively stained preparations of one sample collected at 36 weeks but not in later samples. A polyomavirus was isolated from this sample and also from a sample collected at 39 weeks. No virus particles were detected by electron microscopy in maternal urine post partum, in amniotic fluid at term, in neonatal urine, or in placenta: nor, so far, has a polyomavirus been isolated from any of these specimens. Investigation for cytomegalovirus was also negative. 2.
Corallim, A., and others. Infect. Immun. 1976, 13, 1684.
710 The polyomavirus was JC, a virus previously associated with progressive multifocal leucoencephalopathy and recovered from diseased brain. This is only the third time that JC virus has been isolated from the urine of a patient without neurological disease; the other two had renal transplants and were also investigated by us. The titre of JC heemagglutination-inhibition antibody in maternal serum was 1/1280 at 36 weeks and 1/2560 at 39 weeks’ pregnancy and 5 days post partum. Titres were 1/2560 in the cord blood and 1/1280 in the baby’s serum on day 6. Serum BK antibody levels in the mother and baby were almost identical with the JC antibody titres. Mother and baby went abroad immediately after delivery and are temporarily lost to
follow-up. A polyomavirus has since been cultured from the urine of a second pregnant woman and papovavirus particles have been observed in the urine of three others. Department of Experimental Pathology, St Mary’s Hospital Medical School, London W2 1NY
D. V. COLEMAN R. A. DANIEL
Virus Reference Laboratory, Central Public Health Laboratory, London NW9
S. D. GARDNER A. M. FIELD P. E. GIBSON
THE ANION GAP
SIR,-Your excellent editorial on the anion gapl pays just tribute to the unrivalled paper by Emmett and Narins2 on the clinical value of this calculation. Apart from this aspect I wish to make a plea for the maintenance of this concept in the study of electrolyte balance. Since the sum of cations equals the sum of anions-law of electroneutrality-any discrepancy must be explained. Van Slyke et al. found that the "base" bound by the total plasma-protei ns at pH 7-4’was 0.24 meq/1 for every g/l of protein, thus accounting normally for about 16 meq/1 of anions. The binding of ions to protein is, however, complicated.4 Both normal and abnormal plasma-proteins may contribute to the anions which are not usually measured. Increased protein may reduce the anion gap by attracting cations5 but paraproteins may actually be cationic.6 Greater knowledge of the net charges of normal and abnormal proteins is needed but a very low anion gap may be a useful pointer to
troneutrality. SI units should not be permitted to obscure the importance of the concept of electrolyte balance which has proved to be useful both in diagnosis and in the principles underlying fluid therapy. Department of Chemical Pathology, University of the Witwatersrand Medical School, Johannesburg 2001, South Africa
H. B. STEIN
ALUMINIUM AND ALZHEIMER’S DISEASE
SIR,-There is renewed interest in the neurotoxicity of aluminium compounds, in particular in the aetiology of Alzheimer’s disease. Crapper and co-workers have reportedl,2 that 29% of samples from various regions of the brains of patients dying with Alzheimer’s dementia had raised concentrations of aluminium (>4 t-tg/g dry tissue). We have measured aluminium in 274 brain samples from ten Alzheimer demented (three presenile and seven senile) and nine non-neurological patients. Brains were obtained 3-24 h post mortem and stored at —70°C. Alzheimer’s disease was confirmed histopathologically by assessing the degree of neurofibrillary degeneration in the frontal cortex and hippocampus. Samples of brain (0-3-lg), mainly from the frontal cortex, temporal cortex, hippocampus, corpus callosum, and cerebellum, were analysed for aluminium by atomic-absorption spectroscopy using a dry-ashing procedure.3 Similar numbers of the diseased and the control samples were used from each brain region. ALUMINIUM CONCENTRATIONS IN BRAIN TISSUE
unsuspected multiple myeloma. Without both Cl- and HCO-3 determinations the anion gap be calculated. I agree it is a pity that some laboratories do not report te Cl- in the "routine electrolyte set". Dr Zilva7 is worried about the workload resulting from routine Clrequests. Since most large laboratories possess an automated multichannel analyser perhaps she will one day be persuaded to include the Cl- estimation in her repertoire. Finally, I welcome your "punch line" that electrical charges require expression as meq and not mmol, an important condemnation of SI units in electrolyte studies. The routine set of electrolytes is fortunately univalent, but how is one to record the net cation equivalence of the normal plasma proteins in SI units? Gamble’s8 cation-anion diagrams may involve "a number of assumptions"9 in determining the net equivalence of proteins,4 but they are still useful in providing an insight into the composition of sera based on the law of elec-
cannot
...
1. Lancet, 1977, i, 785. 2. Emmett, M., Narins, R. G. Medicine, 1977, 56, 38. 3. Van Slyke, D. D., Hastings, A. B., Hiller, A., Sendroy Jr.
J. J. biol. Chem. 1928, 79, 769. 4. Van Leeuwen, A. M.Acta med. scand. 1964, 176, (suppl. 422), 1. 5. Gutman, A. B., Gutman, E. B., Jillson, R., Williams, R. D. J. clin. Invest. 1936, 15, 475. 6. Murray, T., Long, W., Narins, R. G. New Engl.J. Med. 1975, 292, 574. 7. Zilva, J. F. Lancet, 1977, i, 948. 8. Gamble, J. L. Chemical Anatomy, Physiology and Pathology of Extracellular Fluid. Cambridge, Mass., 1954. 9. Latner, A. L. Cantarow and Trumper’s Clinical Biochemistry; p. 349. Phila-
delphia, 1975.
*[J-g aluminium/g dry tissueis.E.M.; no. of samples in parentheses. There was no significant difference in aluminium concentration between nine normal brains and ten brains from patients with dementia, in the material as a whole or in the regions rich in neurofibrillary degeneration (hippocampus and frontal and temporal cortex). 21% of senile-dementia samples have raised aluminium concentrations as defined previously,’ but so did 21% of age-matched control samples. This suggests that high aluminium levels are related to age rather than dementia. In contrast to the findings of Crapper et al. we found only a small number of samples with raised aluminium in the presenile dementia but this could be due to the limited number of samples and brain regions investigated in this group. The density of neurofibrillary tangles in the brains of Alzheimer patients is 6-40 times that in brains from mentally normal age-matched controls.4 Crapper et al. suggested that the increase in aluminium concentration correlates well with the extent of neurofibrillary degeneration.2 When we compared all the samples from brains of age 70 years or more we found that 1. 2. 3. 4.
Crapper, D. R., Krishnan, S. S., Dalton, A. J. Science, 1973, 180, 511. Crapper, D. R., Krishnan, S. S., Quittkat, S. Brain, 1976, 99, 67. McDermott, J. R., Whitehill, I. Analyt. chim. Acta, 1976, 85, 195. Ball, M. J.Neuropath. appl. Neurobiol.1976, 2, 395.
indirect immunofluorescence and anticomplement immunofluorescence for the presence of T antibody. All the sera were reacted against a battery of cell lines that included the NMK line nude-mouse kidney cells transformed by BK virus, the VA-2 line (human fibroblast transformed by SV40), and the WI-38 line (diploid human fibroblasts). Of 113 sera tested (see table) 6 had non-specific antinuclear antibodies and 12 gave weak reactions with the viral-transformed cells at a 1/2 dilution. The 12 weakly positive sera were further tested by anticomplement methods at higher dilutions. None of these 12 sera was positive by the anticomplement method. The low titres of reactivity and the lack of complement fixation argue against a specific antigen-antibody reaction at the low dilution in the initial screening. Corallini et al .2 have reported that 11 patients with tumours out of 952 examined had antibodies to BK virus T antigen. Had we examined more sera we also might have found some cancer patients with positive reactions. Nevertheless, although the possibility that human papovaviruses may be associated with a small number of tumours cannot be ruled out, our data suggest a lack of widespread association of these viruses with human neoplasia.
Gusthuis, Amsterdam, Netherlands
Laboratory of Pathology, Division of Cancer Biology and Diagnosis,
Onze Lieve Vrouwen
of Biochemistry,
Department Rijksuniversiteit Limburg
J. M.
VAN
DOORM
H. C. HEMKER
National Cancer Institute,
Bethesda, Maryland 20014, U.S.A.
J. COSTA C. YEE A. S. RABSON
POLYOMA VIRUS IN URINE DURING PREGNANCY
ABSENCE OF PAPOVAVIRUS T ANTIBODY IN PATIENTS WITH MALIGNANCIES
SiR,—JC virus and BK virus, the two prototype viruses of the human polyoma group, are ubiquitous.’ They infect large numbers of people, and in most cases seroconversion takes place in childhood. The ability of these viruses to transform cells in vitro and to produce tumours in laboratory animals has led to suggestions that they may play a role in the aetiology of human neoplasms. ANTIBODIES TO VIRAL T ANTIGENS IN PATIENTS WITH TUMOURS OR BENIGN CONDITIONS AND CONTROLS
Animals bearing tumours induced by BK or JC virus have antibodies to a viral antigen present in the nucleus of the tumour cell. These "T" antigens and antibodies can be detected by immunofluorescence and by complement fixation; BK and JC virus T antigens high degree of cross-reactivity with each other and with the T antigen ofSV40 virus. If any human tumours are caused by BK or JC virus some patients should have antibody to the T antigens of these viruses in their sera. We have tested a panel of human sera by 8. Suttie, J. W. Science, 1973, 179, 192. 9.van Oosterom, A. T., Mattie, H., Hermens, W. Th., Veltkamp, J. J. Thrombos. Hœm. 1976,35,607. 10 van der Meer, J., Hemker, H. C., Loeliger, E. A. Thrombos. Diathes. hœmorrh. 1968, suppl. 19, 1. 1.Padgett, B.L., Walker, D. L. Progr. med. Virol. 1976, 22, 1.
SIR,-Active infection with the human polyomaviruses BK and JC has been recorded only in patients whose immunological responses have been impaired. Our attempts to isolate these viruses from the urinary tract, throat washings, or faeces of children under the age of 15 years who seem immunologically competent have been unsuccessful, despite the fact that antibody studies have shown that most people have been exposed to these viruses and that primary infection occurs most frequently in childhood. In November, 1976, we started a prospective study of 1000 patients at St Mary’s Hospital. We chose pregnant women because of their apparent susceptibility to virus infection, perhaps due to physiological changes in pregnancy. We isolated human polyomavirus JC from the urinary tract of a 38-year-old woman who came to the antenatal clinic on Dec. 12, 1976, in the 36th week of her third pregnancy. Her obstetric history included a spontaneous abortion at 7 weeks in 1972 and the delivery of a normal full-term female infant in 1974. Her third pregnancy proceeded uneventfully to 41 weeks when a normal female infant weighing 3200 g was delivered by spontaneous vertex delivery. The placenta weighed 520 g and appeared normal, as did the cord and membranes. Jaundice developed in the baby after 36 h, and the serum-bilirubin was 176 fLmol/1 rising to 220 fLmol/1 on the sixth day. No evidence of rhesus incompatibility or other cause for the jaundice was found, and the jaundice settled after phototherapy. Maternal infection with a human polyomavirus was suspected as a result of routine light microscopy of stained smears of urinary sediment. A few atypical urothelial cells with large basophilic intranuclear inclusions were observed in cytological smears prepared from urine samples collected at 36, 38, 39, 40, and 41 weeks of pregnancy. A small number of papovavirus particles were observed under the electron microscope in negatively stained preparations of one sample collected at 36 weeks but not in later samples. A polyomavirus was isolated from this sample and also from a sample collected at 39 weeks. No virus particles were detected by electron microscopy in maternal urine post partum, in amniotic fluid at term, in neonatal urine, or in placenta: nor, so far, has a polyomavirus been isolated from any of these specimens. Investigation for cytomegalovirus was also negative. 2.
Corallim, A., and others. Infect. Immun. 1976, 13, 1684.
710 The polyomavirus was JC, a virus previously associated with progressive multifocal leucoencephalopathy and recovered from diseased brain. This is only the third time that JC virus has been isolated from the urine of a patient without neurological disease; the other two had renal transplants and were also investigated by us. The titre of JC heemagglutination-inhibition antibody in maternal serum was 1/1280 at 36 weeks and 1/2560 at 39 weeks’ pregnancy and 5 days post partum. Titres were 1/2560 in the cord blood and 1/1280 in the baby’s serum on day 6. Serum BK antibody levels in the mother and baby were almost identical with the JC antibody titres. Mother and baby went abroad immediately after delivery and are temporarily lost to
follow-up. A polyomavirus has since been cultured from the urine of a second pregnant woman and papovavirus particles have been observed in the urine of three others. Department of Experimental Pathology, St Mary’s Hospital Medical School, London W2 1NY
D. V. COLEMAN R. A. DANIEL
Virus Reference Laboratory, Central Public Health Laboratory, London NW9
S. D. GARDNER A. M. FIELD P. E. GIBSON
THE ANION GAP
SIR,-Your excellent editorial on the anion gapl pays just tribute to the unrivalled paper by Emmett and Narins2 on the clinical value of this calculation. Apart from this aspect I wish to make a plea for the maintenance of this concept in the study of electrolyte balance. Since the sum of cations equals the sum of anions-law of electroneutrality-any discrepancy must be explained. Van Slyke et al. found that the "base" bound by the total plasma-protei ns at pH 7-4’was 0.24 meq/1 for every g/l of protein, thus accounting normally for about 16 meq/1 of anions. The binding of ions to protein is, however, complicated.4 Both normal and abnormal plasma-proteins may contribute to the anions which are not usually measured. Increased protein may reduce the anion gap by attracting cations5 but paraproteins may actually be cationic.6 Greater knowledge of the net charges of normal and abnormal proteins is needed but a very low anion gap may be a useful pointer to
troneutrality. SI units should not be permitted to obscure the importance of the concept of electrolyte balance which has proved to be useful both in diagnosis and in the principles underlying fluid therapy. Department of Chemical Pathology, University of the Witwatersrand Medical School, Johannesburg 2001, South Africa
H. B. STEIN
ALUMINIUM AND ALZHEIMER’S DISEASE
SIR,-There is renewed interest in the neurotoxicity of aluminium compounds, in particular in the aetiology of Alzheimer’s disease. Crapper and co-workers have reportedl,2 that 29% of samples from various regions of the brains of patients dying with Alzheimer’s dementia had raised concentrations of aluminium (>4 t-tg/g dry tissue). We have measured aluminium in 274 brain samples from ten Alzheimer demented (three presenile and seven senile) and nine non-neurological patients. Brains were obtained 3-24 h post mortem and stored at —70°C. Alzheimer’s disease was confirmed histopathologically by assessing the degree of neurofibrillary degeneration in the frontal cortex and hippocampus. Samples of brain (0-3-lg), mainly from the frontal cortex, temporal cortex, hippocampus, corpus callosum, and cerebellum, were analysed for aluminium by atomic-absorption spectroscopy using a dry-ashing procedure.3 Similar numbers of the diseased and the control samples were used from each brain region. ALUMINIUM CONCENTRATIONS IN BRAIN TISSUE
unsuspected multiple myeloma. Without both Cl- and HCO-3 determinations the anion gap be calculated. I agree it is a pity that some laboratories do not report te Cl- in the "routine electrolyte set". Dr Zilva7 is worried about the workload resulting from routine Clrequests. Since most large laboratories possess an automated multichannel analyser perhaps she will one day be persuaded to include the Cl- estimation in her repertoire. Finally, I welcome your "punch line" that electrical charges require expression as meq and not mmol, an important condemnation of SI units in electrolyte studies. The routine set of electrolytes is fortunately univalent, but how is one to record the net cation equivalence of the normal plasma proteins in SI units? Gamble’s8 cation-anion diagrams may involve "a number of assumptions"9 in determining the net equivalence of proteins,4 but they are still useful in providing an insight into the composition of sera based on the law of elec-
cannot
...
1. Lancet, 1977, i, 785. 2. Emmett, M., Narins, R. G. Medicine, 1977, 56, 38. 3. Van Slyke, D. D., Hastings, A. B., Hiller, A., Sendroy Jr.
J. J. biol. Chem. 1928, 79, 769. 4. Van Leeuwen, A. M.Acta med. scand. 1964, 176, (suppl. 422), 1. 5. Gutman, A. B., Gutman, E. B., Jillson, R., Williams, R. D. J. clin. Invest. 1936, 15, 475. 6. Murray, T., Long, W., Narins, R. G. New Engl.J. Med. 1975, 292, 574. 7. Zilva, J. F. Lancet, 1977, i, 948. 8. Gamble, J. L. Chemical Anatomy, Physiology and Pathology of Extracellular Fluid. Cambridge, Mass., 1954. 9. Latner, A. L. Cantarow and Trumper’s Clinical Biochemistry; p. 349. Phila-
delphia, 1975.
*[J-g aluminium/g dry tissueis.E.M.; no. of samples in parentheses. There was no significant difference in aluminium concentration between nine normal brains and ten brains from patients with dementia, in the material as a whole or in the regions rich in neurofibrillary degeneration (hippocampus and frontal and temporal cortex). 21% of senile-dementia samples have raised aluminium concentrations as defined previously,’ but so did 21% of age-matched control samples. This suggests that high aluminium levels are related to age rather than dementia. In contrast to the findings of Crapper et al. we found only a small number of samples with raised aluminium in the presenile dementia but this could be due to the limited number of samples and brain regions investigated in this group. The density of neurofibrillary tangles in the brains of Alzheimer patients is 6-40 times that in brains from mentally normal age-matched controls.4 Crapper et al. suggested that the increase in aluminium concentration correlates well with the extent of neurofibrillary degeneration.2 When we compared all the samples from brains of age 70 years or more we found that 1. 2. 3. 4.
Crapper, D. R., Krishnan, S. S., Dalton, A. J. Science, 1973, 180, 511. Crapper, D. R., Krishnan, S. S., Quittkat, S. Brain, 1976, 99, 67. McDermott, J. R., Whitehill, I. Analyt. chim. Acta, 1976, 85, 195. Ball, M. J.Neuropath. appl. Neurobiol.1976, 2, 395.
