Cytochemical Identification of Tetrahydrofolate in Humar\ Leukocytes LAWRENCE KASS, M.D.
Kass, Lawrence, Cytochemical identification of tetrahydrofolate in human leukocytes. Am J Clin Pathol 71: 574-577, 1979. Utilizing the ability of molybdenum dioxide dichloride to form a black insoluble precipitate with tetrahydrofolate, a cytochemical test for rapid detection of tetrahydrofolate in human leukocytes was developed. The test may be useful in distinguishing deficiency of vitamin Bl2 or folate or both from other disorders of erythropoiesis characterized by megaloblastic erythropoiesis. (Key words: Tetrahydrofolate; Leukocytes.)
5-methyltetrahydrofolate + homocysteine
ability of a molybdenum salt to form a black precipitate with tetrahydrofolate. Materials and Methods Recent cytochemical studies have focused upon identification of substances involved in the following reaction catalyzed by 5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase 2 - 48 :
methylcobalamin-dependent methyltransferase > FADH,
In one of these cytochemical tests, methionine was identified by a bacterial overlay technic. 5 In another test, homocysteine was identified by its ability to form a brown precipitate with nickel salts." As indicated above, the reaction catalyzed by methylcobalamin methyltransferase is also important in generation of tetrahydrofolate from 5-methyltetrahydrofolate. 2,4a In the present study, efforts have been made to ascertain whether tetrahydrofolate, like homocysteine, might have unusual reactivity with heavy metal salts, and if so, whether a cytochemical test for tetrahydrofolate might be developed. Demonstration of presence or absence of tetrahydrofolate could be useful in the rapid assessment of deficiency of vitamin B, 2 or folate. Based on the "methyltetrahydrofolate-trap" hypothesis, 2 ' 4 - 9 intracellular levels of tetrahydrofolate might be expected to be especially low in disorders such as pernicious anemia, in which activity of methylcobalamin methyltransferase and subsequent conversion of 5-methyltetrahydrofolate to Received February 14, 1978; received revised manuscript March 29, 1978. Supported by grants from National Leukemia Association and the Children's Leukemia Foundation of Michigan. Address reprint requests to Dr. Kass: Department of Internal Medicine (Simpson Memorial Institute), University of Michigan, Ann Arbor, Michigan 48109.
methionine + tetrahydrofolate
tetrahydrofolate are greatly reduced compared with normal values. Along these lines, a freshly prepared 1% aqueous solution of tetrahydrofolate* stored in mercaptoethanol to retard decomposition was added to individual 1% aqueous solutions of a wide variety of heavy metal salts. Fortuituously, it was observed that when a 1% solution of molybdenum dioxide dichloride,t p H 1.6, was added to tetrahydrofolate, a dense black percipitate formed. As a result of this observation, other commercially available folates and folate derivatives that could be readily prepared in the laboratory were tested for reactivity with the molybdenum salt. These folates included 1% aqueous solutions of folic acid,* folinic acid (10-formyltetrahydrofolate),* 5-methyltetrahydrofolate,* dihydrofolic acid,* and 5,10-methylenetetrahydrofolate. 8 In the case of folic acid, the solution became yellow after addition of molybdenum salt, and a solution of dihydrofolic acid became light brown. In neither instance was a precipitate visible, either grossly or under the light microscope. Various aspects of the reaction between tetrahydrofolate and molybdenum salt were investigated. It was * Sigma Chemical Co., St. Louis, Missouri. t Alfa, Danvers, Massachusetts.
0002-9173/79/0500/0574 $00.70 © American Society of Clinical Pathologists
574
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
A CYTOCHEMICAL TEST for tetrahydrofolate in human leukocytes is described. The test utilizes the
Department of Internal Medicine (Simpson Memorial Institute), The University of Michigan, Ann Arbor, Michigan
Vol.71 • No. 5
LABORATORY SUGGESTIONS
To further evaluate the specificity of the reaction for tetrahydrofolate, a wide variety of other biologic substances was tested in 1% aqueous solutions. These substances included all of the 21 essential amino acids, and methylated amino acids including NG monomethylL-arginine, NG-dimethyl-L-arginine, N°,N' G -dimethylL-arginine, L-1-methylhistidine, L-3-methylhistidine, and N-epsilon-methyl-L-lysine. In the case of the methylated arginines that were yellow due to the dye dj-p-hydroxyazobenzene-p'-sulfonatemonohydrate, the yellow dye was removed from the amino acid by passage of an aqueous solution of the amino acid through a column of Dowex I. Chromatography of the effluent on Whatman # 1 filter paper in an ascending system using isoproterenol:H 2 0/l N HC1 160:40:8, v/v/v, gave single discrete ninhydrin-positive spots with anticipated Rf values for the respective amino acid. Additional compounds tested included homocysteine thiolactone, glutathione, D-glucose-1-phosphate, Dfructose-6-phosphate, xylulose-5-phosphate, glucose6-phosphate, D-ribulose-5' phosphate, D-erythrose-4phosphate, D-sedoheptulose-7-phosphate, NADP, NADPH, thiamine monophosphate, adenosine diphosphate (ADP), 3'5' cyclic adenosine monophosphate (AMP), 6-phosphogluconic acid, pyridoxyl-5'-phosphate, adenosine-5'-monophosphate, adenosine-5'-triphosphate, isocitric dehydrogenase, citrate synthase, aconitase, malic dehydrogenase, fumarase, glucose-6phosphate dehydrogenase, Co A, S-acetyl Co A, methylmalonyl CoA, succinyl CoA, cocarboxylase, n-propit New England Nuclear, N. Billerica, Massachusetts. § Climax Molybdynum Company, Ann Arbor, Michigan.
onyl CoA, citric acid, methylmalonic acid, succinic acid, L-malic acid, cis-aconitic acid, cisoxaloacetic acid, pyruvic acid, propionic acid, DL-isocitric acid, fumaric acid, oxalosuccinic acid, nonanoic acid, tricosanoic acid heneicosanoic acid, nonadecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanoic acid, undecanoic acid, heptanoic acid, alpha-ketoglutaric acid, flavin adenine dinucleotide (FAD), cytochrome C, 0.1% human serum albumin, calf thymus histone, calf thymus deoxyribonucleic acid (DNA), beta-nicotinamide adenine dinucleotide (NAD), betadiphosphopyridine nucleotide (DPN), ascorbic acid, urea, sucrose, glycogen (rabbit liver), T-coliphage RNA, /3-amylase, ferritin, L-a-lysophosphatidylcholine (egg yolk), methylcobalamin, cyanocobalamin, thymidine monophosphate, s-adenosyl-L-methionine, succinic thiokinase, insulin (bovine), aconitase, L-o-methylthreonine, L-o-methylphenylalanine, L-o-methyltyrosine, L-o-methylserine, fibrinogen, pepsin, trypsin, serotonin, heparin, intrinsic factor, and flavin mononucleotide. Purified alpha and beta chains of human hemoglobin were obtained through the courtesy of Dr. William Winter, Department of Human Genetics, University of Michigan. A wide variety of heavy metal salts was also tested for reactivity with homocysteine, including zirconium, yttrium, silver, copper, barium, lead, cadmium, platinum, gold, calcium, aluminum, vanadium, and various anions, including CO:t , S 0 4 = , CI", r , OH~, and POy in their respective potassium and sodium salts. Of these, methylcobalamin formed an orange-brown color after addition of the molybdenum solution, and serotonin formed a light gray precipitate. White precipitates formed after addition of molybdenum solution to L-histidine, L-arginine, L-3-methylhistidine, thiamine monophosphate, coenzyme A, cocarboxylase, pentadecanoic acid, human serum albumin, intrinsic factor, and beta-amylase. Based upon the seemingly unique reaction of molybdenum with tetrahydrofolate to form a dense black precipitate, a cytochemical test for tetrahydrofolate was developed, recognizing that other yet untested and perhaps unidentified substances may give similar color reactions. Since the test may have greatest applicability in conditions where intracellular concentrations of tetrahydrofolate might be expected to be unusually low, such as deficiency of vitamin B, 2 , patients who had untreated pernicious anemia were utilized, and healthy individuals served as controls. In patients with pernicious anemia, peripheral-blood leukocytes were also obtained 30 days after treatment with vitamin B 12 . Patients with untreated folate deficiency were not available for study. Twenty-milliliter samples of peripheral venous blood
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
found that concentrations of less than 1% molybdenum dioxide dichloride did not form a precipitate with 1% tetrahydrofolate. A 1% solution of molybdenum salt formed a visible precipitate with concentrations of tetrahydrofolate as low as 6 x 10~4 M in the test tube. The black precipitate formed by combining aqueous solutions of molybdenum salt and tetrahydrofolate was soluble in N,N dimethylformamide, and insoluble in distilled water, acetic acid, 1% NaOH, 10% NaOH, 1% HC1, 10% HC1, acetone, methanol, carbon tetrachloride, phenol, toluene, and acetone. To analyze the composition of the precipitate and to determine whether it contained molybdenum and tetrahydrofolate, the precipitate was washed five times in double-distilled water, air-dried, dissolved in N,N dimethylformamide, and analyzed for tetrahydrofolate by radioisotopic dilution method,$ and for molybdenum by atomic absorption technics.§ Analysis showed that the precipitate was composed of molybdenum and tetrahydrofolate in the molar ratio of 3:1.
575
A.J.C.P. • May 1979
K.ASS
576 4 .
IS
^f
, 4 t **-£' •..«• ^
..
v**
••**«•
Results In the samples of blood from healthy persons, buffy-coat leukocytes (polymorphonuclear leukocytes, monocytes, eosinophils, basophils, and lymphocytes) contained small black particles dispersed throughout the cytoplasm (Fig. 1). Particles were particularly numerous in polymorphonuclear leukocytes and less numerous in erythrocytes, lymphocytes, and monocytes (Fig. 1). In samples from patients who had untreated pernicious anemia, black particles were not observed in buffy-coat leukocytes or erythrocytes (Fig. 2). In samples from patients who had treated pernicious anemia, black particles were seen in leukocytes. Black particles were not observed in platelets from either patients or healthy persons.
t
r=* 0. >
*** 1
1 '
«
FIG. 1. Black particles in polymorphonuclear leukocytes from peripheral venous blood of a healthy person. Molybdenum dioxide dichloride. x 1,500.
A cytochemical method for rapid detection of what appears to be tetrahydrofolate in human leukocytes has been outlined. A black precipitate formed when tetrahydrofolate reacted with a molybdenum salt, and served as the basis for the cytochemical test. Among various commercially available and readily synthesized folate derivatives that were tested for reactivity with molybdenum, only tetrahydrofolate formed a black precipitate. Based upon these and other biologic substances tested, the reaction appears to show selectivity for tetrahydrofolate monoglutamate, recognizing that tetrahydrofolate polyglutamate, not readily available for testing, is the intracellular form of tetrahydro-
were obtained in heparinized Vacutainer tubes from four individuals who had untreated pernicious anemia and from eight healthy persons. The tubes were centrifuged at 1,000 x g for 5 min in a Clay-Adams angle-head centrifuge,11 and supernatant plasma was discarded. Buffy coat was removed and washed twice in phosphate-buffered saline solution, pH 7.1.1 To the buffy coat was added 1.0 ml of a freshly prepared solution of molybdenum dioxide dichloride. The solution was gently agitated every 15 sec for a total of 1 min. At the end of this time, the tube containing buffy coat and molybdenum solution was centrifuged at 1,000 x g for 2 min, and the supernatant was discarded. The buffycoat-molybdenum mixture appeared as a brown sludge. Several drops of buffy coat were applied to methanolcleaned glass coverslips, and films were made between the coverslips and air-dried. Without counterstaining, the films were mounted facedown by the use of immersion oil on methanol-cleaned glass slides and were examined under the light microscope. r
Clay-Adams, Parsippany, New Jersey.
FIG. 2. Absence of black particles in a macropolymorphonuclear leukocyte from peripheral venous blood of a patient with untreated pernicious anemia. Molybdenum dioxide dichloride. x 1,500.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Discussion
Vol. 71 . No. 5
LABORATORY SUGGESTIONS
patients with untreated folate deficiency were not available for study. However, the test may be useful as an additional cytochemical screening method for rapid identification of vitamin B12 or folate deficiency states7 and their distinction from other disorders of erythropoiesis in which megaloblasts may be found, such as erythroleukemia and erythremic myelosis.33 References 1. Black MM, Ansley HR: Antigen-induced changes in lymphoid cell histones. I. Thymus. J Cell Biol 26:201-209. 1965 2. Herbert V: Recent developments in cobalamin metabolism. The Cobalamins. Edited by Arnstein HRV, Wrighton RJ. Edinburgh, Churchill-Livingstone, 1971, pp 2-10 3. Kass L, Schnitzer B: Refractory Anemia. Springfield, Charles C Thomas, 1975 4. Kass L: Pernicious Anemia. Philadelphia, W. B. Saunders, 1976 5. Kass L: Detection of methionine in pernicious anemia megaloblasts and other types of erythroid precursors. Am J Clin Pathol 65:504-507, 1976 6. Kass L: Cytochemical identification of homocysteine in pernicious anemia and chronic erythremic myelosis. Am J Clin Pathol 67:53-56, 1977 7. Onicescu D, Popescu M, Pa§ca L: The in situ localization of folic acid. Histochem J 7:427-434, 1975 8. Roberts D: An isotopic assay for thymidylate synthetase. Biochemistry 5:3546-3548, 1966 9. Taylor RT, Hanna ML, Hutton JJ: 5-Methyltetrahydrofolate homocysteine cobalamin methyltransferase in human bone marrow and its relationship in pernicious anemia. Arch Biochem Biophys 165:787-795, 1974
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
folate. Possibly, other substances not yet tested, including other folate derivatives such as 5,10-methynyltetrahydrofolate, may also form colored precipitates with molybdenum. Reasons for tetrahydrofolate's having the property of forming a black precipitate with molybdenum have not been elucidated. In addition to test-tube precipitations, several other observations support the postulate that black particles in leukocytes reacted with molybdenum represent sites of combination of tetrahydrofolate with this heavy metal. First, black particles were absent in leukocytes from patients untreated pernicious anemia, a disorder in which intracellular concentrations of tetrahydrofolate are believed to be greatly reduced.2-49 Second, in samples of blood from patients with treated pernicious anemia, black particles appeared in leukocytes. These findings suggest that in untreated pernicious anemia, an intracellular metabolite was either present in amounts too small to be detected by molybdenum or virtually absent. After treatment with vitamin B,2, the metabolite (presumably tetrahydrofolate) reappeared in amounts sufficiently large to react visibly with molybdenum salt. Probably the test will not distinguish between a deficiency of vitamin B12 and a deficiency of folate, since in the latter condition, intracellular concentrations of tetrahydrofolate may also be low. In the present study, this postulate could not be tested, since
577
Kass, Lawrence, Cytochemical identification of tetrahydrofolate in human leukocytes. Am J Clin Pathol 71: 574-577, 1979. Utilizing the ability of molybdenum dioxide dichloride to form a black insoluble precipitate with tetrahydrofolate, a cytochemical test for rapid detection of tetrahydrofolate in human leukocytes was developed. The test may be useful in distinguishing deficiency of vitamin Bl2 or folate or both from other disorders of erythropoiesis characterized by megaloblastic erythropoiesis. (Key words: Tetrahydrofolate; Leukocytes.)
5-methyltetrahydrofolate + homocysteine
ability of a molybdenum salt to form a black precipitate with tetrahydrofolate. Materials and Methods Recent cytochemical studies have focused upon identification of substances involved in the following reaction catalyzed by 5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase 2 - 48 :
methylcobalamin-dependent methyltransferase > FADH,
In one of these cytochemical tests, methionine was identified by a bacterial overlay technic. 5 In another test, homocysteine was identified by its ability to form a brown precipitate with nickel salts." As indicated above, the reaction catalyzed by methylcobalamin methyltransferase is also important in generation of tetrahydrofolate from 5-methyltetrahydrofolate. 2,4a In the present study, efforts have been made to ascertain whether tetrahydrofolate, like homocysteine, might have unusual reactivity with heavy metal salts, and if so, whether a cytochemical test for tetrahydrofolate might be developed. Demonstration of presence or absence of tetrahydrofolate could be useful in the rapid assessment of deficiency of vitamin B, 2 or folate. Based on the "methyltetrahydrofolate-trap" hypothesis, 2 ' 4 - 9 intracellular levels of tetrahydrofolate might be expected to be especially low in disorders such as pernicious anemia, in which activity of methylcobalamin methyltransferase and subsequent conversion of 5-methyltetrahydrofolate to Received February 14, 1978; received revised manuscript March 29, 1978. Supported by grants from National Leukemia Association and the Children's Leukemia Foundation of Michigan. Address reprint requests to Dr. Kass: Department of Internal Medicine (Simpson Memorial Institute), University of Michigan, Ann Arbor, Michigan 48109.
methionine + tetrahydrofolate
tetrahydrofolate are greatly reduced compared with normal values. Along these lines, a freshly prepared 1% aqueous solution of tetrahydrofolate* stored in mercaptoethanol to retard decomposition was added to individual 1% aqueous solutions of a wide variety of heavy metal salts. Fortuituously, it was observed that when a 1% solution of molybdenum dioxide dichloride,t p H 1.6, was added to tetrahydrofolate, a dense black percipitate formed. As a result of this observation, other commercially available folates and folate derivatives that could be readily prepared in the laboratory were tested for reactivity with the molybdenum salt. These folates included 1% aqueous solutions of folic acid,* folinic acid (10-formyltetrahydrofolate),* 5-methyltetrahydrofolate,* dihydrofolic acid,* and 5,10-methylenetetrahydrofolate. 8 In the case of folic acid, the solution became yellow after addition of molybdenum salt, and a solution of dihydrofolic acid became light brown. In neither instance was a precipitate visible, either grossly or under the light microscope. Various aspects of the reaction between tetrahydrofolate and molybdenum salt were investigated. It was * Sigma Chemical Co., St. Louis, Missouri. t Alfa, Danvers, Massachusetts.
0002-9173/79/0500/0574 $00.70 © American Society of Clinical Pathologists
574
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
A CYTOCHEMICAL TEST for tetrahydrofolate in human leukocytes is described. The test utilizes the
Department of Internal Medicine (Simpson Memorial Institute), The University of Michigan, Ann Arbor, Michigan
Vol.71 • No. 5
LABORATORY SUGGESTIONS
To further evaluate the specificity of the reaction for tetrahydrofolate, a wide variety of other biologic substances was tested in 1% aqueous solutions. These substances included all of the 21 essential amino acids, and methylated amino acids including NG monomethylL-arginine, NG-dimethyl-L-arginine, N°,N' G -dimethylL-arginine, L-1-methylhistidine, L-3-methylhistidine, and N-epsilon-methyl-L-lysine. In the case of the methylated arginines that were yellow due to the dye dj-p-hydroxyazobenzene-p'-sulfonatemonohydrate, the yellow dye was removed from the amino acid by passage of an aqueous solution of the amino acid through a column of Dowex I. Chromatography of the effluent on Whatman # 1 filter paper in an ascending system using isoproterenol:H 2 0/l N HC1 160:40:8, v/v/v, gave single discrete ninhydrin-positive spots with anticipated Rf values for the respective amino acid. Additional compounds tested included homocysteine thiolactone, glutathione, D-glucose-1-phosphate, Dfructose-6-phosphate, xylulose-5-phosphate, glucose6-phosphate, D-ribulose-5' phosphate, D-erythrose-4phosphate, D-sedoheptulose-7-phosphate, NADP, NADPH, thiamine monophosphate, adenosine diphosphate (ADP), 3'5' cyclic adenosine monophosphate (AMP), 6-phosphogluconic acid, pyridoxyl-5'-phosphate, adenosine-5'-monophosphate, adenosine-5'-triphosphate, isocitric dehydrogenase, citrate synthase, aconitase, malic dehydrogenase, fumarase, glucose-6phosphate dehydrogenase, Co A, S-acetyl Co A, methylmalonyl CoA, succinyl CoA, cocarboxylase, n-propit New England Nuclear, N. Billerica, Massachusetts. § Climax Molybdynum Company, Ann Arbor, Michigan.
onyl CoA, citric acid, methylmalonic acid, succinic acid, L-malic acid, cis-aconitic acid, cisoxaloacetic acid, pyruvic acid, propionic acid, DL-isocitric acid, fumaric acid, oxalosuccinic acid, nonanoic acid, tricosanoic acid heneicosanoic acid, nonadecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanoic acid, undecanoic acid, heptanoic acid, alpha-ketoglutaric acid, flavin adenine dinucleotide (FAD), cytochrome C, 0.1% human serum albumin, calf thymus histone, calf thymus deoxyribonucleic acid (DNA), beta-nicotinamide adenine dinucleotide (NAD), betadiphosphopyridine nucleotide (DPN), ascorbic acid, urea, sucrose, glycogen (rabbit liver), T-coliphage RNA, /3-amylase, ferritin, L-a-lysophosphatidylcholine (egg yolk), methylcobalamin, cyanocobalamin, thymidine monophosphate, s-adenosyl-L-methionine, succinic thiokinase, insulin (bovine), aconitase, L-o-methylthreonine, L-o-methylphenylalanine, L-o-methyltyrosine, L-o-methylserine, fibrinogen, pepsin, trypsin, serotonin, heparin, intrinsic factor, and flavin mononucleotide. Purified alpha and beta chains of human hemoglobin were obtained through the courtesy of Dr. William Winter, Department of Human Genetics, University of Michigan. A wide variety of heavy metal salts was also tested for reactivity with homocysteine, including zirconium, yttrium, silver, copper, barium, lead, cadmium, platinum, gold, calcium, aluminum, vanadium, and various anions, including CO:t , S 0 4 = , CI", r , OH~, and POy in their respective potassium and sodium salts. Of these, methylcobalamin formed an orange-brown color after addition of the molybdenum solution, and serotonin formed a light gray precipitate. White precipitates formed after addition of molybdenum solution to L-histidine, L-arginine, L-3-methylhistidine, thiamine monophosphate, coenzyme A, cocarboxylase, pentadecanoic acid, human serum albumin, intrinsic factor, and beta-amylase. Based upon the seemingly unique reaction of molybdenum with tetrahydrofolate to form a dense black precipitate, a cytochemical test for tetrahydrofolate was developed, recognizing that other yet untested and perhaps unidentified substances may give similar color reactions. Since the test may have greatest applicability in conditions where intracellular concentrations of tetrahydrofolate might be expected to be unusually low, such as deficiency of vitamin B, 2 , patients who had untreated pernicious anemia were utilized, and healthy individuals served as controls. In patients with pernicious anemia, peripheral-blood leukocytes were also obtained 30 days after treatment with vitamin B 12 . Patients with untreated folate deficiency were not available for study. Twenty-milliliter samples of peripheral venous blood
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
found that concentrations of less than 1% molybdenum dioxide dichloride did not form a precipitate with 1% tetrahydrofolate. A 1% solution of molybdenum salt formed a visible precipitate with concentrations of tetrahydrofolate as low as 6 x 10~4 M in the test tube. The black precipitate formed by combining aqueous solutions of molybdenum salt and tetrahydrofolate was soluble in N,N dimethylformamide, and insoluble in distilled water, acetic acid, 1% NaOH, 10% NaOH, 1% HC1, 10% HC1, acetone, methanol, carbon tetrachloride, phenol, toluene, and acetone. To analyze the composition of the precipitate and to determine whether it contained molybdenum and tetrahydrofolate, the precipitate was washed five times in double-distilled water, air-dried, dissolved in N,N dimethylformamide, and analyzed for tetrahydrofolate by radioisotopic dilution method,$ and for molybdenum by atomic absorption technics.§ Analysis showed that the precipitate was composed of molybdenum and tetrahydrofolate in the molar ratio of 3:1.
575
A.J.C.P. • May 1979
K.ASS
576 4 .
IS
^f
, 4 t **-£' •..«• ^
..
v**
••**«•
Results In the samples of blood from healthy persons, buffy-coat leukocytes (polymorphonuclear leukocytes, monocytes, eosinophils, basophils, and lymphocytes) contained small black particles dispersed throughout the cytoplasm (Fig. 1). Particles were particularly numerous in polymorphonuclear leukocytes and less numerous in erythrocytes, lymphocytes, and monocytes (Fig. 1). In samples from patients who had untreated pernicious anemia, black particles were not observed in buffy-coat leukocytes or erythrocytes (Fig. 2). In samples from patients who had treated pernicious anemia, black particles were seen in leukocytes. Black particles were not observed in platelets from either patients or healthy persons.
t
r=* 0. >
*** 1
1 '
«
FIG. 1. Black particles in polymorphonuclear leukocytes from peripheral venous blood of a healthy person. Molybdenum dioxide dichloride. x 1,500.
A cytochemical method for rapid detection of what appears to be tetrahydrofolate in human leukocytes has been outlined. A black precipitate formed when tetrahydrofolate reacted with a molybdenum salt, and served as the basis for the cytochemical test. Among various commercially available and readily synthesized folate derivatives that were tested for reactivity with molybdenum, only tetrahydrofolate formed a black precipitate. Based upon these and other biologic substances tested, the reaction appears to show selectivity for tetrahydrofolate monoglutamate, recognizing that tetrahydrofolate polyglutamate, not readily available for testing, is the intracellular form of tetrahydro-
were obtained in heparinized Vacutainer tubes from four individuals who had untreated pernicious anemia and from eight healthy persons. The tubes were centrifuged at 1,000 x g for 5 min in a Clay-Adams angle-head centrifuge,11 and supernatant plasma was discarded. Buffy coat was removed and washed twice in phosphate-buffered saline solution, pH 7.1.1 To the buffy coat was added 1.0 ml of a freshly prepared solution of molybdenum dioxide dichloride. The solution was gently agitated every 15 sec for a total of 1 min. At the end of this time, the tube containing buffy coat and molybdenum solution was centrifuged at 1,000 x g for 2 min, and the supernatant was discarded. The buffycoat-molybdenum mixture appeared as a brown sludge. Several drops of buffy coat were applied to methanolcleaned glass coverslips, and films were made between the coverslips and air-dried. Without counterstaining, the films were mounted facedown by the use of immersion oil on methanol-cleaned glass slides and were examined under the light microscope. r
Clay-Adams, Parsippany, New Jersey.
FIG. 2. Absence of black particles in a macropolymorphonuclear leukocyte from peripheral venous blood of a patient with untreated pernicious anemia. Molybdenum dioxide dichloride. x 1,500.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Discussion
Vol. 71 . No. 5
LABORATORY SUGGESTIONS
patients with untreated folate deficiency were not available for study. However, the test may be useful as an additional cytochemical screening method for rapid identification of vitamin B12 or folate deficiency states7 and their distinction from other disorders of erythropoiesis in which megaloblasts may be found, such as erythroleukemia and erythremic myelosis.33 References 1. Black MM, Ansley HR: Antigen-induced changes in lymphoid cell histones. I. Thymus. J Cell Biol 26:201-209. 1965 2. Herbert V: Recent developments in cobalamin metabolism. The Cobalamins. Edited by Arnstein HRV, Wrighton RJ. Edinburgh, Churchill-Livingstone, 1971, pp 2-10 3. Kass L, Schnitzer B: Refractory Anemia. Springfield, Charles C Thomas, 1975 4. Kass L: Pernicious Anemia. Philadelphia, W. B. Saunders, 1976 5. Kass L: Detection of methionine in pernicious anemia megaloblasts and other types of erythroid precursors. Am J Clin Pathol 65:504-507, 1976 6. Kass L: Cytochemical identification of homocysteine in pernicious anemia and chronic erythremic myelosis. Am J Clin Pathol 67:53-56, 1977 7. Onicescu D, Popescu M, Pa§ca L: The in situ localization of folic acid. Histochem J 7:427-434, 1975 8. Roberts D: An isotopic assay for thymidylate synthetase. Biochemistry 5:3546-3548, 1966 9. Taylor RT, Hanna ML, Hutton JJ: 5-Methyltetrahydrofolate homocysteine cobalamin methyltransferase in human bone marrow and its relationship in pernicious anemia. Arch Biochem Biophys 165:787-795, 1974
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
folate. Possibly, other substances not yet tested, including other folate derivatives such as 5,10-methynyltetrahydrofolate, may also form colored precipitates with molybdenum. Reasons for tetrahydrofolate's having the property of forming a black precipitate with molybdenum have not been elucidated. In addition to test-tube precipitations, several other observations support the postulate that black particles in leukocytes reacted with molybdenum represent sites of combination of tetrahydrofolate with this heavy metal. First, black particles were absent in leukocytes from patients untreated pernicious anemia, a disorder in which intracellular concentrations of tetrahydrofolate are believed to be greatly reduced.2-49 Second, in samples of blood from patients with treated pernicious anemia, black particles appeared in leukocytes. These findings suggest that in untreated pernicious anemia, an intracellular metabolite was either present in amounts too small to be detected by molybdenum or virtually absent. After treatment with vitamin B,2, the metabolite (presumably tetrahydrofolate) reappeared in amounts sufficiently large to react visibly with molybdenum salt. Probably the test will not distinguish between a deficiency of vitamin B12 and a deficiency of folate, since in the latter condition, intracellular concentrations of tetrahydrofolate may also be low. In the present study, this postulate could not be tested, since
577
