1008
Letters to the Editor
mentally retarded." We apologize for overlooking his subsequent statement that at the time o f examination she appeared to be normally intelligent, though unable to read or write, presumably of inadequate education. He states that Case 3 "was considered mentally retarded" without subsequent clarification o f her intelligence. That patient, however, was not available for biochemical study and the diagnosis of galactokinase deficiency was made on probability in view o f the family gene. In regard to Dr. Gitzelmann's second point, we did not catalogue a "normal human trait" in a large number o f subjects. We compiled a list of three biochemical and three clinical findings in the small group of 12 previously published patients with galactokinase deficiency, and suggest that their frequency suggests these may be manifestations o f the spectrum of this disorder. Clearly, only when many more patients are identified will this be known. For the present, it would not be wise to assume that galactokinase deficiency is a benign disorder other than involvement o f the lens. Nathan Litman, M.D. Montefiore Hospital and Medical Center Albert Einstein College o f Medicine Bronx, hr. Y. REFERENCES
1. Litman N, Kanter A, and Finberg L: Galactokinase deficiency presenting as pseudotumor cerebri, J PEDIATR 86:410, 1975. 2. Gitzelmann R: Hereditary galactokinase deficiency, a newly recognized cause of juvenile cataracts, Pediatr Res 1:14, 1967.
The Journal of Pediatrics December 1975
REFERENCES
1. Swick HM: Calcification of intervertebral discs in childhood, J PEDIATR 86:364, 1975. 2. Sutton TJ, and Turcotte B: Posterior herniation of calcified 9intervertebral discs in children, J Can Assoc Radiol 24:131, 1973. 3. Peck FC: A calcified thoracic intervertebral disk with herniation and spinal cord compression in a child, J Neurosurg 14:105, 1957.
Reply To the Editor: I appreciate the opportunity to reply to the letter from Tudor Sutton, M.D., regarding my paper, "Calcification of intervertebral discs in childhood." Dr. Sutton is quite correct in stating that most children with disc calcification do not require specific therapy, particularly neurosurgical intervention. When a child presents, however, with signs of acute spinal cord compression, it is necessary to establish a diagnosis quickly, since permanent spinal cord damage may ensue from significant extrinsic compression of any etiology within only a few hours. If neuologic signs are minimal and if myelography shows only minimal compression of the spinal cord, conservative therapy with careful observation of the child's neurologic condition might be warranted. If signs of advancing deficit become apparent, however, surgery may well become necessary. H. M. Swick, M.D., F.A.A.P. Assistant Professor of Neurology and Pediatrics University of Kentucky Lexington, Ky. 40506
Calcification of intervertebral discs in childhood To the Editor: I was very interested by the article "Calcification of intervertebral discs in childhood, TM describing two children who underwent neurosurgical intervention for signs of nerve root or spinal cord compression. We have published two similar cases-' with documented regression of clinical and myelographic signs of compression under conservative management. This stands to reason when one considers the description at surgery given bS~ Swick and by Peck:' of soft disc material. We therefore do not feel neurosurgical intervention is recommended even in patients with signs of nerve root or spinal cord compression. Of course, if compression with severe neurological deficit were present, neurosurgery would be indicated. This, however, does not seem to occur in this condition. Tudor J. Sutton, M.D., F.R.C.P.(C) 185, rue du Chevaleret 75013 Paris, France
Determination of bilirubin-binding capacity of serum albumin To the Editor: I should like to comment about the paper "Fluorescent dye method for the determination of the bilirubin binding capacity of serum albumin" by Lee, Gartner, and Zarafu ~which appeared in the February issue of TH~ JOURNAL OF PEDIATRICS. A basic assumption is made both in the presentation of the results and the discussion that the decrease in fluorescence of the Duct Yellow 7 (DY7) dye is due to its displacement from the albumin molecule. No direct proof is given that this is the case, nor is there any proof that the bilirubin displaces the dye from binding to the same site on the albumin. It appears to be another indirect technique for determining bilirubin-binding capacity similar to that of the HABBA dye mentioned in their discussion. No direct correlation is given between the AF and serum
Letters to the Editor
Volume 87 Number 6, part 1
bilirubin concentrations in order to determine binding capacity as performed in a routine clinical situation. The change in the fluorescence of the DY7 dye at pH values below 7.4 does not necessarily indicate that less dye is being bound. It could also indicate changes in the ionization of the dye and/or on the protein molecule where the dye is bound, which could influence its ability to absorb and emit radiation. No data are given on the fluorescence property of the DY7 as a function of pH in the absence of serum. Since bilirubin has a broad absorption spectra encompassing both the activation at 390 nm and emission at 430 nm, one would expect that a filter effect could result in the decrease in fluorescence. Fluorescence quenched by the bilirubin would be proportional to the concentration of bilirubin. The amount of quench, due to bilirubin, would be different in the absence of protein from that in the presence of the protein. Quench would be proportional to the amount of fluorescence: the greater the fluorescence the greater the actual fluorescence change for the same concentration of bilirubin. Determination of the quench in the absence of serum would result in a lower quantitative change which cannot be applied directly to the fluorescence that might be quenched in the presence of protein. The break in the curve that is shown in Fig. 6 is rather similar to that observed when the optical density at 460 nm of a serum sample is measured as a function of the bilirubin concentration. If the inner filter effect were operating, the slight shift in the absorbance would result in a shift of the fluorescence quench as well. No mention at all is made of the fluorescence that is produced by bilirubin when bound to albumin. This fluorescence is produced by the direct interaction of the bilirubin with the albumin molecule and does not require the addition of another substance to complicate the observation. What we do not need is another indirect method to determine bilirubin-binding capacity, since the literature is already confused in this regard. 2-6 Joseph Krasner, Ph.D. Children "s Hospital Buffalo, N.Y. 14222 Colin F. Chignell, Ph.D. National Heart and Lung Institute Bethesda, Md. 20014
REFERENCES
1. Lee K, Gariner LM, and Zarafu I: Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin, J PEDIATR86:280, 1975. 2. Chen RF: The fluorescence of bilirubin-albumin complexes in fluorescence techniques, in cell biology. Edited by A.A. Thaer, & M. Sernetz. Springer-Verlag, New York, 1973. 3. C h e n RF: Fluorescence stopped flow of relaxation processes in the binding of bilirubin to serum albumin, Arch Biochem Biophys 160:106, 1974. 4. Krasner J: Fluorescent properties of bovine serum albuminbilirubin comples, Biochem Med 7:135, 1973. 5. Kragner J, Giacoia GP, and Yaffe SJ: Drug-protein binding in the newborn infant, Ann N Y Acad Sci 226:101, 1973.
10 0 9
Beaven GH, d'Albis A, and Gratzer WB: The interaction of bilirubin with human serum albumin, Eur J Biochem 33:500, 1973.
Reply To the Editor: Drs. Krasner and Chignell raise a number of theoretical questions regarding the nature of bilirubin binding to albumin in general and the DY7 binding method specifically. The specificity of the binding of the DY7 dye to bilirubinbinding sites on albumin was strongly suggested by the inhibition curve (Fig. 6) presented in the paper. In, as yet unpublished, studies using Sephadex G-200 column chromatographic techniques we have demonstrated competitive displacement of bilirubin from albumin by DY7 at high dye concentrations, supporting the interpretation that the decrease in fluorescence results from displacement of DY7 from its albumin-binding sites by bilirubin. The method presented is, indeed, an indirect technique for determining bilirubin binding, but so is the van den Bergh method for measurement of bilirubin concentrations in serum. This in no way invalidates the usefulness of the technique. It may, in fact, enhance it by increasing sensitivity and accuracy. Direct correlation of AF and clinical interpretations of binding data must await the evaluations currently in progress in our laboratory and neonatal center. The effect of pH on dye binding and fluorescence is complex. As stated in the text, "decreasing the pH of the DY7 solution without added sera led to a marked decrease in the intensity of fluorescence." For reasons of brevity the data were not presented. There is no question that changes in pH may alter the dye structure or its ionization. That in no way alters the significance of the observations, however. The pH curve for the dye in sera was presented to demonstrate the remarkable similarity of pHdependent AF responses to previously published data by other authors on bilirubin binding to albumin. The quench correction curve presented in the paper is based on the fluorescence produced by the DY7 solution prior to addition of seruml The degree of correction is extremely small even at the highest bilirubin concentration used (80 mg/dl). This is so because of enormous dilution of the sample in the buffer containing the dye (1:3,333). Even with higher fluorescence emission (65 units), as seen in solutions equivalent to the enhanced fluorescence of solutions containing serum, the quench is exactly the same. Thus quench is independent of fluorescence emission and dependent only on bilirubin concentration. The quench correction curve given in the paper is applicable at all AF values. The interpretation of the break in the curve in Fig. 6 as being due simply to the concentration of bilirubin independent of binding affinity or capacity is not tenable in view of other data which we have presented elsewhere and which demonstrate a similar break in the curve, but at much lower bilirubin concentrations, in both human cord sera and purified albumin solu-
Letters to the Editor
mentally retarded." We apologize for overlooking his subsequent statement that at the time o f examination she appeared to be normally intelligent, though unable to read or write, presumably of inadequate education. He states that Case 3 "was considered mentally retarded" without subsequent clarification o f her intelligence. That patient, however, was not available for biochemical study and the diagnosis of galactokinase deficiency was made on probability in view o f the family gene. In regard to Dr. Gitzelmann's second point, we did not catalogue a "normal human trait" in a large number o f subjects. We compiled a list of three biochemical and three clinical findings in the small group of 12 previously published patients with galactokinase deficiency, and suggest that their frequency suggests these may be manifestations o f the spectrum of this disorder. Clearly, only when many more patients are identified will this be known. For the present, it would not be wise to assume that galactokinase deficiency is a benign disorder other than involvement o f the lens. Nathan Litman, M.D. Montefiore Hospital and Medical Center Albert Einstein College o f Medicine Bronx, hr. Y. REFERENCES
1. Litman N, Kanter A, and Finberg L: Galactokinase deficiency presenting as pseudotumor cerebri, J PEDIATR 86:410, 1975. 2. Gitzelmann R: Hereditary galactokinase deficiency, a newly recognized cause of juvenile cataracts, Pediatr Res 1:14, 1967.
The Journal of Pediatrics December 1975
REFERENCES
1. Swick HM: Calcification of intervertebral discs in childhood, J PEDIATR 86:364, 1975. 2. Sutton TJ, and Turcotte B: Posterior herniation of calcified 9intervertebral discs in children, J Can Assoc Radiol 24:131, 1973. 3. Peck FC: A calcified thoracic intervertebral disk with herniation and spinal cord compression in a child, J Neurosurg 14:105, 1957.
Reply To the Editor: I appreciate the opportunity to reply to the letter from Tudor Sutton, M.D., regarding my paper, "Calcification of intervertebral discs in childhood." Dr. Sutton is quite correct in stating that most children with disc calcification do not require specific therapy, particularly neurosurgical intervention. When a child presents, however, with signs of acute spinal cord compression, it is necessary to establish a diagnosis quickly, since permanent spinal cord damage may ensue from significant extrinsic compression of any etiology within only a few hours. If neuologic signs are minimal and if myelography shows only minimal compression of the spinal cord, conservative therapy with careful observation of the child's neurologic condition might be warranted. If signs of advancing deficit become apparent, however, surgery may well become necessary. H. M. Swick, M.D., F.A.A.P. Assistant Professor of Neurology and Pediatrics University of Kentucky Lexington, Ky. 40506
Calcification of intervertebral discs in childhood To the Editor: I was very interested by the article "Calcification of intervertebral discs in childhood, TM describing two children who underwent neurosurgical intervention for signs of nerve root or spinal cord compression. We have published two similar cases-' with documented regression of clinical and myelographic signs of compression under conservative management. This stands to reason when one considers the description at surgery given bS~ Swick and by Peck:' of soft disc material. We therefore do not feel neurosurgical intervention is recommended even in patients with signs of nerve root or spinal cord compression. Of course, if compression with severe neurological deficit were present, neurosurgery would be indicated. This, however, does not seem to occur in this condition. Tudor J. Sutton, M.D., F.R.C.P.(C) 185, rue du Chevaleret 75013 Paris, France
Determination of bilirubin-binding capacity of serum albumin To the Editor: I should like to comment about the paper "Fluorescent dye method for the determination of the bilirubin binding capacity of serum albumin" by Lee, Gartner, and Zarafu ~which appeared in the February issue of TH~ JOURNAL OF PEDIATRICS. A basic assumption is made both in the presentation of the results and the discussion that the decrease in fluorescence of the Duct Yellow 7 (DY7) dye is due to its displacement from the albumin molecule. No direct proof is given that this is the case, nor is there any proof that the bilirubin displaces the dye from binding to the same site on the albumin. It appears to be another indirect technique for determining bilirubin-binding capacity similar to that of the HABBA dye mentioned in their discussion. No direct correlation is given between the AF and serum
Letters to the Editor
Volume 87 Number 6, part 1
bilirubin concentrations in order to determine binding capacity as performed in a routine clinical situation. The change in the fluorescence of the DY7 dye at pH values below 7.4 does not necessarily indicate that less dye is being bound. It could also indicate changes in the ionization of the dye and/or on the protein molecule where the dye is bound, which could influence its ability to absorb and emit radiation. No data are given on the fluorescence property of the DY7 as a function of pH in the absence of serum. Since bilirubin has a broad absorption spectra encompassing both the activation at 390 nm and emission at 430 nm, one would expect that a filter effect could result in the decrease in fluorescence. Fluorescence quenched by the bilirubin would be proportional to the concentration of bilirubin. The amount of quench, due to bilirubin, would be different in the absence of protein from that in the presence of the protein. Quench would be proportional to the amount of fluorescence: the greater the fluorescence the greater the actual fluorescence change for the same concentration of bilirubin. Determination of the quench in the absence of serum would result in a lower quantitative change which cannot be applied directly to the fluorescence that might be quenched in the presence of protein. The break in the curve that is shown in Fig. 6 is rather similar to that observed when the optical density at 460 nm of a serum sample is measured as a function of the bilirubin concentration. If the inner filter effect were operating, the slight shift in the absorbance would result in a shift of the fluorescence quench as well. No mention at all is made of the fluorescence that is produced by bilirubin when bound to albumin. This fluorescence is produced by the direct interaction of the bilirubin with the albumin molecule and does not require the addition of another substance to complicate the observation. What we do not need is another indirect method to determine bilirubin-binding capacity, since the literature is already confused in this regard. 2-6 Joseph Krasner, Ph.D. Children "s Hospital Buffalo, N.Y. 14222 Colin F. Chignell, Ph.D. National Heart and Lung Institute Bethesda, Md. 20014
REFERENCES
1. Lee K, Gariner LM, and Zarafu I: Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin, J PEDIATR86:280, 1975. 2. Chen RF: The fluorescence of bilirubin-albumin complexes in fluorescence techniques, in cell biology. Edited by A.A. Thaer, & M. Sernetz. Springer-Verlag, New York, 1973. 3. C h e n RF: Fluorescence stopped flow of relaxation processes in the binding of bilirubin to serum albumin, Arch Biochem Biophys 160:106, 1974. 4. Krasner J: Fluorescent properties of bovine serum albuminbilirubin comples, Biochem Med 7:135, 1973. 5. Kragner J, Giacoia GP, and Yaffe SJ: Drug-protein binding in the newborn infant, Ann N Y Acad Sci 226:101, 1973.
10 0 9
Beaven GH, d'Albis A, and Gratzer WB: The interaction of bilirubin with human serum albumin, Eur J Biochem 33:500, 1973.
Reply To the Editor: Drs. Krasner and Chignell raise a number of theoretical questions regarding the nature of bilirubin binding to albumin in general and the DY7 binding method specifically. The specificity of the binding of the DY7 dye to bilirubinbinding sites on albumin was strongly suggested by the inhibition curve (Fig. 6) presented in the paper. In, as yet unpublished, studies using Sephadex G-200 column chromatographic techniques we have demonstrated competitive displacement of bilirubin from albumin by DY7 at high dye concentrations, supporting the interpretation that the decrease in fluorescence results from displacement of DY7 from its albumin-binding sites by bilirubin. The method presented is, indeed, an indirect technique for determining bilirubin binding, but so is the van den Bergh method for measurement of bilirubin concentrations in serum. This in no way invalidates the usefulness of the technique. It may, in fact, enhance it by increasing sensitivity and accuracy. Direct correlation of AF and clinical interpretations of binding data must await the evaluations currently in progress in our laboratory and neonatal center. The effect of pH on dye binding and fluorescence is complex. As stated in the text, "decreasing the pH of the DY7 solution without added sera led to a marked decrease in the intensity of fluorescence." For reasons of brevity the data were not presented. There is no question that changes in pH may alter the dye structure or its ionization. That in no way alters the significance of the observations, however. The pH curve for the dye in sera was presented to demonstrate the remarkable similarity of pHdependent AF responses to previously published data by other authors on bilirubin binding to albumin. The quench correction curve presented in the paper is based on the fluorescence produced by the DY7 solution prior to addition of seruml The degree of correction is extremely small even at the highest bilirubin concentration used (80 mg/dl). This is so because of enormous dilution of the sample in the buffer containing the dye (1:3,333). Even with higher fluorescence emission (65 units), as seen in solutions equivalent to the enhanced fluorescence of solutions containing serum, the quench is exactly the same. Thus quench is independent of fluorescence emission and dependent only on bilirubin concentration. The quench correction curve given in the paper is applicable at all AF values. The interpretation of the break in the curve in Fig. 6 as being due simply to the concentration of bilirubin independent of binding affinity or capacity is not tenable in view of other data which we have presented elsewhere and which demonstrate a similar break in the curve, but at much lower bilirubin concentrations, in both human cord sera and purified albumin solu-
