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Biotin enrichment in oligodendrocytes in the rat brain
Brain Research, 444 (!988) 199-203
199
Elsevier BRE 22787
Biotin enrichment in oligodendrocytes in the rat brain Steven M. LeVine and Wendy B. Macklin Mental Retardation Research Center, Deparonent of Psychiatry, University of California at Los Angeles. Los Angeles, CA 90024 (U.S.A. )
(Accepted 1 December 1987) Key words: Biotin; Oligodendrocyte; Carbonic anhydrase: Acetyl-coenzyme A carboxylase; Pyruvate carboxylase
Cytochemical localization of endogenous biotin in the rat brain was detected by two different staining methods, avidin-biotin-HRP and goat-anti-biotin with HRP-rabbit-anti-goat. In both staining methods, oligodendrocytes were labeled to a far greater degree than other brain cells. This finding may be important for identification of the role of carbonic anhydrase, which is elevated in the oligodendrocyte, and it may help to explain how the oligodendrocyte generates large quantities of lipids for myelin production.
In recent years, biotin has become a commonly used reagent in cell staining techniques, both for immunocytochemistry and for in situ hybridization. When bound to a probe, e.g. an antibody, lectin or DNA molecule, biotin can be detected by a complex of avidin and biotin conjugated to an enzyme, or by an anti-biotin antibody and an anti-lgG specific second antibody conjugated to an enzyme. The biotinavidin system is quite sensitive, because of the signal enhancement produced by the presence of 4 biotin binding sites per avidin molecule. The biotinylated probes are particularly useful for in situ hybridization studies, because they are non-radioactive probes. Thus, the signal is seen within the cell. rather than as silver grains in an emulsion over the cell. Furthermore, the signal is obtained much more rapidly than that of radioactive probes. However, the current studies suggest some problems associated with staining oligodendrocytes with biotin-labeled probes. The present studies were begun in order to stain sections of brain ti'ssue using biotinylated oligonucleotide probes specific for oligodendrocyte mRNAs, and an avidin-biotin-enzyme complex for visualization. Oligodendrocyte staining was obtained in these experiments, but an equivalent staining pat-
tern was observed in control experiments when the biotinylated probes were eliminated from the staining procedure. We hypothesized that this "non-specific' staining of oligodendrocytes by the avidin-biotin-enzyme complex resulted from the presence of biotin in oligodendrocytes at levels significantly above those found in other brain cells. In this report we validate this hypothesis and discuss the importance of this finding. Sprague-Dawley rats (,Mental Retardation Research Center. UCLA) ranging in age from postnatal day 14 (PI4) to P29 were used. This time frame was selected because it represents the peak of rnyelinogenesis !1. Frozen tissue sections were incubated with an affinity-purified anti-biotin lgG prepared in goats. and selective staining of oligodendrocytes was observed. Oligodendrocytes were seen throughout the entire subcortical white matter. The cells were often observed aligned in rows (Fig. IA. arrowhead; iE). a feature of l_~,~," "'~-~---'~a.',~-~L:mar ....... oligodendrocytes. No cells were observed with the morphology of astrocytes, microglia, or neurons in the subcortical white matter. or in any other region of the brain. When the tissue was incubated with avidin, prior to incubation with anti-biotin the staining of oligodendrocytes was
Correspondence: W.B. Macklin, Mental Retardation Research Center. Department of Psychiatry. University of California at Los
Angeles. Los Angeles. CA 90024. U.S.A. 0(]06-8993/88/$03,50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
200
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Fig. 1. Localization of biotin in oligodendrocytes with anti-biotin. Rats were anesthetized, decapitated and the brains were removed. The brains were placed in a vial and immediately immersed in liquid nitrogen. Twelve/~m coronal sections from the most anterior region of the corpus callosum to the most anterior region of the hippocampus were cut on a cryostat and thaw-mounted on gelatimcoated slides. Slides were stored at -20 °C until use. Slides were used within 1 week of storage. Immunocytochemistry was performed by allowing the slides to come to room temperature and incubating the slides in the following solutions: 4% paraformaldehyde in PBS, 30 rain; PBS x 2, 10 rain; 2% bovine serum albumin (BSA) (Sigma, St. Louis, MO) and 10% normal rabbit serum (Vector Labs., Burlingame, CA) in PBS, pH 7,4 (diluent), overnight, 1:300 goat anti-biotin affinity-purified with biotin (Vector) in diluent, 60-9{) min: PBS x 2, 5 min; 1:25 HRP-labeled rabbit anti-goat IgG (Cooper Biomedical, Malvern, PA) in PBS with 2% BSA, pH 7.4, 60 rain, PBS x 2, 5 min, 1.5 mg 3,3'-diaminobenzidine (DAB; Sigma): 3 ml 0.05 M Tris (Sigma) pH 7.6:1/l130% H20 2 added immediately before use, 8 min in the dark; PBS x 2, 5 min; dehydrated, mounted in Permount (Fisher, Fair Lawn, N J). A-E: subcortical white matter at P29. x426. A: goat anti-biotin staining is observed in oligodendrocytes. Oligodendrocytes are frequently observed aligned in rows (arrowhead). B: the tissue was incubated overnight in 1:50 avidin (Vector) in diluent in place of the normal overnight blocking step prior to incubation with goat anti-biotin. Oligodendrocyte staining is greatly reduced. C: goat anti-biotin was eliminated from the procedure. Oligodendrocyte staining is not observed. D: the biotin antibody was preadsorbed overnight with biotin-labeled agarose (Vector), centrifuged, and incubated with the sections as described above. Oligodendrocyte staining is eliminated. E: sections were heated to 55 °C in PBS prior to incubation with diluent. Anti-biotin staining is seen in oligodendrocytes.
greatly reduced (compare Fig. 1A and 1B). Since biotin is a small molecule (244 D a ) and avidin has a molecular weight of 67,900, the binding of avidin to biotin prior to the incubation with anti-biotin would be expected to block the available sites for an.t_i-biotin. In other control experiments, anti-biotin was either preadsorbed overnight with biotin-conjugated agarose, or it was not included in the staining procedure. In both of these experiments the oligodendrocyte staining was eliminated (Fig. 1C,D). A second set of experiments employing an avidin and biotin-horseradish peroxidase ( H R P ) complex was performed to confirm the enrichment of biotin in oligodendrocytes. As in the earlier experiments, oligodendrocytes were selectively stained (Fig. 2A).
W h e n avidin was eliminated from the p r o c e d u r e or p r e a d s o r b e d against biotin-conjugated agarose~ the oligodendrocyte staining was eliminated (Fig. 2B,C). Besides the subcortical white matter, oligodendrocyte staining was also observed in other white m a t t e r and gray m a t t e r regions of the anterior forebrain. Oligodendrocytes were present in the anterior commissure (Fig. 3A), olfactory tract (Fig. 3B), and in the basal ganglia (Fig. 3C). Cells likely to be oligodendrocytes were also observed in the cortex (Fig. 30). In order to use biotinylated probes in future in situ hybridization studies, it was necessary to establish a protocol to eliminate the binding of avidin or antibiotin to the e n d o g e n o u s biotin present in oligoden-
201
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Fig. 2. Localization of biotin in o!igodendrocytes by avidin-biotin-HRP. Following the fixation step described in Fig. 1, slides were incubated in the following solutions: PBS x 2, 5 min; 2% BSA in PBS, pH 7.4, overnight; 1:100avidin and 1:50 biotin-HRP (Vector), premixed 30 min and incubated 90-120 min; PBS x 2, 5 min; 1.5 mg DAB: 3 ml 0.05 M Tris pH 7.6: I l~130% H20 _,added immediately before use, 8 min in the dark; PBS x 2, 5 min; dehydrated; mounted in Permount. A-D: subcortical white matter at P29. x415. A: oligodendrocyte staining is observed, but it is lighter than that seen with anti-biotin (Fig. 1). B: when avidin was not added in the procedurc, or (C) preadsorbed against biotin-conjugated agarose, the staining was eliminated. D: sections were heated to 55 °C prior to incubation with 2% BSA/PBS. Oligodendrocyte staining is eliminated.
drocytes. Heating sections to 55 °C for 30 min in phosphate-buffered saline (pBS) following fixation with paraformaldehyde eliminated the staining in sections treated with a v i d i n - b i o t i n - H R P (Fig. 2D) but not with anti-biotin (Fig. 1E). Because oligodendrocyte staining is preserved using anti-biotin following heat treatment, we conclude that the absence of staining with a v i d i n - b i o t i n - H R P after heating is not due to the removal of biotin. We suggest instead that heating causes a conformational change in biotin or biotin-linked molecules that prevents the biotin from binding to avidin. In contrast, the antibodies against biotin are a heterogenous population of molecules that bi~d to m~;,ny antigenic epit~pes of biotin. Although heating may prevent some antibodies from reacting with biotin, others clearly do react. The detection of biotin in oligodendrocytes by avidin-biotin-.HRP or by anti-biotin is also affected by different tissue fixations. We found that fixation with 10% acetic acid/90% ethanol or 100% methanol, respectively, eliminated or greatly reduced the ability to detect biotin with either a v i d i n - b i o t i n - H R P or goat anti-biotin (data not shown). Fixation with acetone or 3.7% formaldehyde in PBS preserved the
reactivity of biotin in a similar manner to that seen with 4% paraformaldehyde in PBS. Biotin reactivity with both a v i d i n - b i o t i n - H R P and anti-biotin was eliminated in Faraffin-embedded tissue (data not shown). Thus, endogenous biotin in the brain would not be observed in histocytochemicai studies conducted using certain fixations or paraffin embedding. Biotin is known to be a co-factor for 4 different enzymes in mammals 3. Two of these enzymes, acetyl coenzyme A (CoA) carboxylase and pyruvate carboxylase, generate products that are used in the production of fatty acids -'''~. Acetyl CoA carboxylase catalyzes the carboxylation of acetyl CoA to malonyl CoA, representing the first committed step of fatty acid synthesis TM. Pyruvate carboxylase, a mitochondrial enzyme, is believed to contribute to fatty acid synthesis by a more indirect route than acetyl CoA carboxylase I'-'. Pyruvate carboxylase catalyzes the formation of oxaloacetate from pyruvate. Oxaloacetate then condenses with acetyl CoA to form citrate in order to cross the mitochondrial mcmbrane. Once in the cytosol, citrate breaks down into oxaloacetate and acetyl CoA, and acetyl CoA can be used to generate fatty acids. Thus pyruvate carboxylase ensures
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Fig. 3. Oligodendrocytes enriched in biotin are present in both white and gray matter regions of the brain. Oiigodendrocyte staining was observed with anti-biotin in (A) the anterior commissure, (B) the olfactory tract, and (C) the basal ganglia. D: cells that are likely oligodendrocytes were also observed in the cortex. An identical, but lighter staining pattern to that observed in A through D was observed with avidin-biotin-HRP. A - C P29, x 163. D P29, x326.
normal citric acid cycle activity and the presence of cytosolic citrate for lipogenesis. Forty percent of the total lipid of rat brain is found in myelin ]°. Since myelin is synthesized by the oligodendrocyte, it is not surprising that biotin and/or biotin bound to the enzymes involved in fatty acid synthesis would be significantly increased in the oligodendrocyte over other cell types in the brain. We make the obvious inference that the biotin in oligodendrocytes is predominantly used by the enzyme(s) acetyl CoA carboxylase and/or pyruvate carboxylase and that one or both of these enzymes is increased in oligodendrocytes over other cell types in the brain. It is interesting to view the elevated levels of biotin, and probably biotin-containing enzymes, in oligodendrocytes with respect to other enzymes that are present at elevated levels in oligodendrocytes, particularly
carbonic anhydrase 5-8'n3. Carbonic anhydrase catalyzes the reversible hydration of CO212. It has yet to be determined, however, whether carbonic anhydrase functions in oligodendrocytes to generate CO,. or carbonic acid, i.e., HCO3-. Our data add support to the proposal by Cammer 4 that the carbonic anhydrase equilibrium could favor the generation of HCO 3- for utilization by acetyl CoA carboxylase and pyruvate carboxylase to donate CO., to their respective substrates, acetyl CoA and pyruvate. As further support, it might be noted that the two other biotincontaining enzymes, propionyl-CoA carboxylase and 3-methylcrotonyi-CoA carboxylase also utilize HCO 3- to donate CO2 to their substrates. It is possible that in addition to being attached to enzymes, biotin is in a free or lipid-bound state within oligodendrocytes. This concept is somewhat sup-
203 p o r t e d by the o b s e r v a t i o n that the reactivity of b o t h anti-biotin and avidin-biotin-HRP disappear following fixation with m e t h a n o l or 10% acetic acid/90% ethanol. H o w e v e r , the current data are insufficient to assess the validity of this intriguing possibility since the antigenicity of a given m o l e c u l e , and
1 Achuta Murthy, P.N. and Mistry, S.P., Biotin, Prog. Food Nutr. Sci.. 2 (1977) 405-455. 2 Ballard, F.J. and Hanson, R.W., The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate, J. Lipid Res., 8 (1967) 73-79. 3 Bonjour, J.P., Biotin. In L.J. Machlin (Ed.), Handbook of Vitamins, Nutritional, Biochemical and Clinical Aspects, Dekker, New York, 1984, pp. 403-435. 4 Cammer, W., Oligodendrocyte-associated enzymes. In W.T. Norton (Ed.), Advance~ in Neurochemistry, Vol. 5, Plenum, New York, 1984, pp. 199-232. 5 Cammer, W., Sacchi, R., Kahn, S. and Sapirstein, V., Oligodendroglial structures and distribution shown by carbonic anhydrase immunostaining in the spinal cords of developing normal and shiverer mice, J. Neurosci. Res., 14 (1985) 303-316. 6 Cammer, W., Sacchi, R. and Sapirstein, V., lmmunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (.shiverer) adult mice with comparisons among fixation methods, J. Histochem. Cytochem., 33 (1985) 45-54. 7 Ghandour. M.S., Langley, O.K., Vincedon, G., Gombos, G., Filippi, D., Limozin, N., Dalmasso, C. and Laurent,
possibly its reactivity with avidin, might be differentially affected by different fixations. This w o r k was supported by N I H Grants NS23715, NS25304, HD07032 and a R e s e a r c h C a r e e r Development Award (W.B.M.).
G., lmmunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum: A marker for oligodendrocytes, Neuroscience, 5 (1980) 559-571. 8 Kumpulainen, T. and Nystrom, S.H.M., Immunohistochemical localization of carbonic anhydrase isoenzyme C in human brain, Brain Research, 220 (1981) 220-225. 9 Lynen, G., The role of biotin-dependent carboxylations in biosynthetic reactions, Biochem. J., 102 (1967) 381-400. 10 Morell, P. and Toews, A.D., In vivo metabolism of oligodendrogiial lipids. In W.T. Norton (Ed.), Advances in Neurochemistry, Vol. 5, Plenum, New York, 1984, pp. 47-86. l I Norton, W.T. and Poduslo, S.E., Myelination in rat brain: changes in myelin composition during brain maturation, J. Neurochem., 21 (1973)759-773. 12 Sapirstein, V.S., Carbonic anhydrase. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 4, Plenum, New York, 1983, pp. 385-402. 13 Spicer, S., Stoward, P.J. and Tashian, R.E., The immunohistolocalization of carbonic anhydrase in rodent tissues, J. Histochem. Cytochem., 27 (1979) 820-821. 14 Wakil, S.J., A malonic acid derivative as an intermediate in fatty acid synthesis, Am. ChenL Soc. J., 80 (1958) 6465.
199
Elsevier BRE 22787
Biotin enrichment in oligodendrocytes in the rat brain Steven M. LeVine and Wendy B. Macklin Mental Retardation Research Center, Deparonent of Psychiatry, University of California at Los Angeles. Los Angeles, CA 90024 (U.S.A. )
(Accepted 1 December 1987) Key words: Biotin; Oligodendrocyte; Carbonic anhydrase: Acetyl-coenzyme A carboxylase; Pyruvate carboxylase
Cytochemical localization of endogenous biotin in the rat brain was detected by two different staining methods, avidin-biotin-HRP and goat-anti-biotin with HRP-rabbit-anti-goat. In both staining methods, oligodendrocytes were labeled to a far greater degree than other brain cells. This finding may be important for identification of the role of carbonic anhydrase, which is elevated in the oligodendrocyte, and it may help to explain how the oligodendrocyte generates large quantities of lipids for myelin production.
In recent years, biotin has become a commonly used reagent in cell staining techniques, both for immunocytochemistry and for in situ hybridization. When bound to a probe, e.g. an antibody, lectin or DNA molecule, biotin can be detected by a complex of avidin and biotin conjugated to an enzyme, or by an anti-biotin antibody and an anti-lgG specific second antibody conjugated to an enzyme. The biotinavidin system is quite sensitive, because of the signal enhancement produced by the presence of 4 biotin binding sites per avidin molecule. The biotinylated probes are particularly useful for in situ hybridization studies, because they are non-radioactive probes. Thus, the signal is seen within the cell. rather than as silver grains in an emulsion over the cell. Furthermore, the signal is obtained much more rapidly than that of radioactive probes. However, the current studies suggest some problems associated with staining oligodendrocytes with biotin-labeled probes. The present studies were begun in order to stain sections of brain ti'ssue using biotinylated oligonucleotide probes specific for oligodendrocyte mRNAs, and an avidin-biotin-enzyme complex for visualization. Oligodendrocyte staining was obtained in these experiments, but an equivalent staining pat-
tern was observed in control experiments when the biotinylated probes were eliminated from the staining procedure. We hypothesized that this "non-specific' staining of oligodendrocytes by the avidin-biotin-enzyme complex resulted from the presence of biotin in oligodendrocytes at levels significantly above those found in other brain cells. In this report we validate this hypothesis and discuss the importance of this finding. Sprague-Dawley rats (,Mental Retardation Research Center. UCLA) ranging in age from postnatal day 14 (PI4) to P29 were used. This time frame was selected because it represents the peak of rnyelinogenesis !1. Frozen tissue sections were incubated with an affinity-purified anti-biotin lgG prepared in goats. and selective staining of oligodendrocytes was observed. Oligodendrocytes were seen throughout the entire subcortical white matter. The cells were often observed aligned in rows (Fig. IA. arrowhead; iE). a feature of l_~,~," "'~-~---'~a.',~-~L:mar ....... oligodendrocytes. No cells were observed with the morphology of astrocytes, microglia, or neurons in the subcortical white matter. or in any other region of the brain. When the tissue was incubated with avidin, prior to incubation with anti-biotin the staining of oligodendrocytes was
Correspondence: W.B. Macklin, Mental Retardation Research Center. Department of Psychiatry. University of California at Los
Angeles. Los Angeles. CA 90024. U.S.A. 0(]06-8993/88/$03,50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
200
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B
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D
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Fig. 1. Localization of biotin in oligodendrocytes with anti-biotin. Rats were anesthetized, decapitated and the brains were removed. The brains were placed in a vial and immediately immersed in liquid nitrogen. Twelve/~m coronal sections from the most anterior region of the corpus callosum to the most anterior region of the hippocampus were cut on a cryostat and thaw-mounted on gelatimcoated slides. Slides were stored at -20 °C until use. Slides were used within 1 week of storage. Immunocytochemistry was performed by allowing the slides to come to room temperature and incubating the slides in the following solutions: 4% paraformaldehyde in PBS, 30 rain; PBS x 2, 10 rain; 2% bovine serum albumin (BSA) (Sigma, St. Louis, MO) and 10% normal rabbit serum (Vector Labs., Burlingame, CA) in PBS, pH 7,4 (diluent), overnight, 1:300 goat anti-biotin affinity-purified with biotin (Vector) in diluent, 60-9{) min: PBS x 2, 5 min; 1:25 HRP-labeled rabbit anti-goat IgG (Cooper Biomedical, Malvern, PA) in PBS with 2% BSA, pH 7.4, 60 rain, PBS x 2, 5 min, 1.5 mg 3,3'-diaminobenzidine (DAB; Sigma): 3 ml 0.05 M Tris (Sigma) pH 7.6:1/l130% H20 2 added immediately before use, 8 min in the dark; PBS x 2, 5 min; dehydrated, mounted in Permount (Fisher, Fair Lawn, N J). A-E: subcortical white matter at P29. x426. A: goat anti-biotin staining is observed in oligodendrocytes. Oligodendrocytes are frequently observed aligned in rows (arrowhead). B: the tissue was incubated overnight in 1:50 avidin (Vector) in diluent in place of the normal overnight blocking step prior to incubation with goat anti-biotin. Oligodendrocyte staining is greatly reduced. C: goat anti-biotin was eliminated from the procedure. Oligodendrocyte staining is not observed. D: the biotin antibody was preadsorbed overnight with biotin-labeled agarose (Vector), centrifuged, and incubated with the sections as described above. Oligodendrocyte staining is eliminated. E: sections were heated to 55 °C in PBS prior to incubation with diluent. Anti-biotin staining is seen in oligodendrocytes.
greatly reduced (compare Fig. 1A and 1B). Since biotin is a small molecule (244 D a ) and avidin has a molecular weight of 67,900, the binding of avidin to biotin prior to the incubation with anti-biotin would be expected to block the available sites for an.t_i-biotin. In other control experiments, anti-biotin was either preadsorbed overnight with biotin-conjugated agarose, or it was not included in the staining procedure. In both of these experiments the oligodendrocyte staining was eliminated (Fig. 1C,D). A second set of experiments employing an avidin and biotin-horseradish peroxidase ( H R P ) complex was performed to confirm the enrichment of biotin in oligodendrocytes. As in the earlier experiments, oligodendrocytes were selectively stained (Fig. 2A).
W h e n avidin was eliminated from the p r o c e d u r e or p r e a d s o r b e d against biotin-conjugated agarose~ the oligodendrocyte staining was eliminated (Fig. 2B,C). Besides the subcortical white matter, oligodendrocyte staining was also observed in other white m a t t e r and gray m a t t e r regions of the anterior forebrain. Oligodendrocytes were present in the anterior commissure (Fig. 3A), olfactory tract (Fig. 3B), and in the basal ganglia (Fig. 3C). Cells likely to be oligodendrocytes were also observed in the cortex (Fig. 30). In order to use biotinylated probes in future in situ hybridization studies, it was necessary to establish a protocol to eliminate the binding of avidin or antibiotin to the e n d o g e n o u s biotin present in oligoden-
201
B.
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: 2,i::: )
Fig. 2. Localization of biotin in o!igodendrocytes by avidin-biotin-HRP. Following the fixation step described in Fig. 1, slides were incubated in the following solutions: PBS x 2, 5 min; 2% BSA in PBS, pH 7.4, overnight; 1:100avidin and 1:50 biotin-HRP (Vector), premixed 30 min and incubated 90-120 min; PBS x 2, 5 min; 1.5 mg DAB: 3 ml 0.05 M Tris pH 7.6: I l~130% H20 _,added immediately before use, 8 min in the dark; PBS x 2, 5 min; dehydrated; mounted in Permount. A-D: subcortical white matter at P29. x415. A: oligodendrocyte staining is observed, but it is lighter than that seen with anti-biotin (Fig. 1). B: when avidin was not added in the procedurc, or (C) preadsorbed against biotin-conjugated agarose, the staining was eliminated. D: sections were heated to 55 °C prior to incubation with 2% BSA/PBS. Oligodendrocyte staining is eliminated.
drocytes. Heating sections to 55 °C for 30 min in phosphate-buffered saline (pBS) following fixation with paraformaldehyde eliminated the staining in sections treated with a v i d i n - b i o t i n - H R P (Fig. 2D) but not with anti-biotin (Fig. 1E). Because oligodendrocyte staining is preserved using anti-biotin following heat treatment, we conclude that the absence of staining with a v i d i n - b i o t i n - H R P after heating is not due to the removal of biotin. We suggest instead that heating causes a conformational change in biotin or biotin-linked molecules that prevents the biotin from binding to avidin. In contrast, the antibodies against biotin are a heterogenous population of molecules that bi~d to m~;,ny antigenic epit~pes of biotin. Although heating may prevent some antibodies from reacting with biotin, others clearly do react. The detection of biotin in oligodendrocytes by avidin-biotin-.HRP or by anti-biotin is also affected by different tissue fixations. We found that fixation with 10% acetic acid/90% ethanol or 100% methanol, respectively, eliminated or greatly reduced the ability to detect biotin with either a v i d i n - b i o t i n - H R P or goat anti-biotin (data not shown). Fixation with acetone or 3.7% formaldehyde in PBS preserved the
reactivity of biotin in a similar manner to that seen with 4% paraformaldehyde in PBS. Biotin reactivity with both a v i d i n - b i o t i n - H R P and anti-biotin was eliminated in Faraffin-embedded tissue (data not shown). Thus, endogenous biotin in the brain would not be observed in histocytochemicai studies conducted using certain fixations or paraffin embedding. Biotin is known to be a co-factor for 4 different enzymes in mammals 3. Two of these enzymes, acetyl coenzyme A (CoA) carboxylase and pyruvate carboxylase, generate products that are used in the production of fatty acids -'''~. Acetyl CoA carboxylase catalyzes the carboxylation of acetyl CoA to malonyl CoA, representing the first committed step of fatty acid synthesis TM. Pyruvate carboxylase, a mitochondrial enzyme, is believed to contribute to fatty acid synthesis by a more indirect route than acetyl CoA carboxylase I'-'. Pyruvate carboxylase catalyzes the formation of oxaloacetate from pyruvate. Oxaloacetate then condenses with acetyl CoA to form citrate in order to cross the mitochondrial mcmbrane. Once in the cytosol, citrate breaks down into oxaloacetate and acetyl CoA, and acetyl CoA can be used to generate fatty acids. Thus pyruvate carboxylase ensures
202
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Fig. 3. Oligodendrocytes enriched in biotin are present in both white and gray matter regions of the brain. Oiigodendrocyte staining was observed with anti-biotin in (A) the anterior commissure, (B) the olfactory tract, and (C) the basal ganglia. D: cells that are likely oligodendrocytes were also observed in the cortex. An identical, but lighter staining pattern to that observed in A through D was observed with avidin-biotin-HRP. A - C P29, x 163. D P29, x326.
normal citric acid cycle activity and the presence of cytosolic citrate for lipogenesis. Forty percent of the total lipid of rat brain is found in myelin ]°. Since myelin is synthesized by the oligodendrocyte, it is not surprising that biotin and/or biotin bound to the enzymes involved in fatty acid synthesis would be significantly increased in the oligodendrocyte over other cell types in the brain. We make the obvious inference that the biotin in oligodendrocytes is predominantly used by the enzyme(s) acetyl CoA carboxylase and/or pyruvate carboxylase and that one or both of these enzymes is increased in oligodendrocytes over other cell types in the brain. It is interesting to view the elevated levels of biotin, and probably biotin-containing enzymes, in oligodendrocytes with respect to other enzymes that are present at elevated levels in oligodendrocytes, particularly
carbonic anhydrase 5-8'n3. Carbonic anhydrase catalyzes the reversible hydration of CO212. It has yet to be determined, however, whether carbonic anhydrase functions in oligodendrocytes to generate CO,. or carbonic acid, i.e., HCO3-. Our data add support to the proposal by Cammer 4 that the carbonic anhydrase equilibrium could favor the generation of HCO 3- for utilization by acetyl CoA carboxylase and pyruvate carboxylase to donate CO., to their respective substrates, acetyl CoA and pyruvate. As further support, it might be noted that the two other biotincontaining enzymes, propionyl-CoA carboxylase and 3-methylcrotonyi-CoA carboxylase also utilize HCO 3- to donate CO2 to their substrates. It is possible that in addition to being attached to enzymes, biotin is in a free or lipid-bound state within oligodendrocytes. This concept is somewhat sup-
203 p o r t e d by the o b s e r v a t i o n that the reactivity of b o t h anti-biotin and avidin-biotin-HRP disappear following fixation with m e t h a n o l or 10% acetic acid/90% ethanol. H o w e v e r , the current data are insufficient to assess the validity of this intriguing possibility since the antigenicity of a given m o l e c u l e , and
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possibly its reactivity with avidin, might be differentially affected by different fixations. This w o r k was supported by N I H Grants NS23715, NS25304, HD07032 and a R e s e a r c h C a r e e r Development Award (W.B.M.).
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