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Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity
13
Cfiriica Chintica Acru, 129 (1983) 13-18 Elsevier Biomedical Press
CCA 2439
Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity Gillian
P. Smith
a,*, Barbara
B. Anderson
’ and Timothy
J. Peters
a
u Division oj Clinical Cell Biolop, MRC Clinical Research Centre, Harrow, Middlesex (lJK) und ’ Department
of Haematology,
St. Barthoiemew’s (Received
Hospiral,
West Smithfiefd,
London (UK)
August 24th. 1982)
Summary Neutrophil leukocytes. isolated from normal subjects and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation, showed very similar cytosol distributions of ‘pyridoxal and pyridoxal phosphate and lactate dehydrogenase. The small amounts of pyridoxal and pyridoxal phosphate associated with the dense granule fractions were not associated with the alkaline phosphatase containing granules. The levels of pyridoxal and pyridoxal phosphate were determined in neutrophils from control subjects, women in the third trimester of pregnancy and patients with chronic granulocytic leukaemia. Neutrophil pyridoxal phosphate was increased in women in the third trimester of pregnancy compared to controls, but there was little variation in the level of pyridoxal between the groups. There was no consistent correlation between the pyridoxal phosphate and the neutrophil alkaline phosphatase activity in the patient groups, Although in vitro neutrophil alkaline phosphatase rapidly hydrolyses pyridoxal phosphate, it is suggested that in vivo this is unlikely to be the principal function of the enzyme.
Introduction Pyridoxal 5’-phosphate, the coenzyme form of pyridoxine (vitamin $) has an important function in numerous biochemical reactions. The pyridoxal content of tissues is probably regulated by a number of factors such as plasma membrane
* Correspondence: Watford Road,
0009-898
Dr. G.P. Smith, Division of Clinical Harrow, Middlesex, HA1 3UJ, UK.
I /83/0000-0000/$03.00
0 1983 Else&x
Cell Biology,
Science Publishers
MRC Clinical
Research
Centre.
14
transport [l], by phosphorylation of free pyridoxal [2], and by binding of the coenzyme to protein [3,4]. It has also been shown that cellular phosphatases (EC 3.1.3.-) play an important role in regulating tissue pyridoxal phosphate levels [5]. When the amount of pyridoxal phosphate exceeds the binding capacity of the tissue apoproteins, free pyridoxai phosphate is rapidfy hydrolysed and the released pyridoxal can readily traverse the cell membrane. Normal human polymorphonuclear leukocytes are a rich source of alkaline phosphatase and the levels of activity vary considerably in certain neutrophil disorders and in pregnancy [6,7]. This alkaline phosphatase activity has been localised to a unique organelle (phosphasome) of the neutrophil [8,9], although the physiological function of this organelle is, as yet, unclear. Recently, analytical subcellular fractionation experiments IlO] and EM cytochemical studies [ 1l] have clearly shown that alkaline pyridoxal phosphate phosphatase is also located to the phosphasome granules. In addition to sharing the same subcellular localisation both alkaline pyridoxal phosphate phosphatase and alkaline phosphatase activities shared identical pH optima and Mg”+ requirements, and showed similar inhibition curves with levamisole, a specific inhibitor ,of alkaline phosphatase. Since the specific activities of the enzymes in neutrophils isolated from women in late pregnancy and patients with chronic granulocytic leukaemia varied in a parallel manner when compared to controls, it was suggested that pyridoxal phosphate may be a physiological substrate for neutrophil alkaline phosphatase [IO]. The present study reports the subcellular localisation of pyridoxal and pyridoxal phosphate in human neutrophils using analytical subcellular fractionation techniques and compares the levels of these vitamers in chronic granulocytic leukaemia and pregnancy, conditions known to exhibit striking variations in alkaline phosphatase and alkaline pyridoxal phosphate phosphatase levels compared with control subjects. Materials and methods Preparation of human polymorphon~c~ear leukocytes Polymorphonuclear leukocytes were isolated by dextran sedimentation and Ficoll-Hypaque centrifugation as previously described [7]. Blood, 50 ml, was used directly from control subjects and pregnant women, but blood from leukaemic patients, with a high leukocyte count, was diluted with an equal volume of 0.15 mol/l NaCI. Levels of pyridoxa( and pyridoxal phosphate The neutrophils were resuspended in 4.5 ml ice-cold 0.2 mol/l Na acetate-acetic acid buffer, pH 4.0, and disrupted in a Dounce homogeniser by 30 strokes of a tight-fitting (Type B) pestle. A portion was removed for protein determination and the remainder boiled for 3 min. For the estimation of free pyridoxal 2 ml boiled neutrophils were mixed with 1 ml 0.2 mol/I Na acetate buffer, pH 4.0, and I ml 1.2 mol/I trichloroacetic acid. The mixture was heated at SO’C for 5 min to ensure complete precipitation of the protein. After centrifugation at 800 x g for 5 min the
15
supernatant was removed and retained for analysis. For total pyridoxal 2 ml boiled neutrophils were added ta I ml 0.2 m&,/l Na acetate-acetic acid buffer, pH 4.0, containing 0.34 mU acid phosphatase (potato Type II, Sigma London Ltd. London, UK) and incubated at 37°C for 60 min. This procedure hydrolysed all pyridoxal phosphate present to pyridoxal. After cooling 1 ml 1.2 mol/l trichloroacetic acid was added and the samples treated exactly as those for the estimation of free pyridoxal. The level of pyridoxal in the extracts was estimated with a modification of the fluorimetric method of Takanashi et al ]12].
Subeeklar
locaiisation ofpyridoxal and pyridoxal phosphate
The isolated neutrophils were pelleted in 4.5 ml 0.2 mol/l sucrose medium containing 1 mmol/l Na, EDTA, pH 7.2, and 5000 units heparin/I, and disrupted in a Dounce homogeniser as described previously [13]. The homogenate was centrifuged at 800 x g for 10 min and the post-nuclear su~ernatant removed, stored on ice and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation [14]. ApproximateIy 5 ml of the postnuclear supernatant was Iayered onto a 28 ml sucrose density gradient, extending linearly with respect to volume, from a density of 1.05 g * crK’ to one of 1.28 g. cmV3 and resting on a 6 ml cushion of density 1.32 g - cm -’ in a Beaufay automatic zonal rotor. All gradient solutions contained 1 mmol/t Na, EDTA pH 7.2, and 5000 units heparin/l and were prepared in double distilled deionised water which had been passed through a Millipore filter. The rotor was run at 35000 rev/min for 3.5 min and, after slowing to 8000 rev/min, some 15 fractions were collected, weighed and their density determined as described previously (141. Part of each fraction and the post-nuclear supernatant was retained for enzymic analysis, and the remainder heated in a boihng water bath for 5 min. One ml of each fraction was then treated and extracted in the same way as the cell homogenates, prior to estimation of free and total pyridoxal. After centrifugation the supernatants were extracted with 3 x 8 ml of ether and the final extract made up to a suitable volume with deionised water. The Level of pyridoxal was determined by the microbiological method of Anderson et al ]15], using ~ctobar~~~us casei as assay organism. The difference between the total and free pyridoxal represented pyridoxal phosphate. Corrections were made for the trace levels of pyridoxal and pyridoxai phosphate contents of the sucrose density gradient solutions. Patient groups studied were as reported previously [7]. Results
~~bcet~ular ~oca~~~ation of ~~r~doxai and ~~rjdoxa~ phosphate Fig. 1 shows the mean density gradient distribution of pyridoxal phosphate and some of the organelle marker enzymes of the human distribution profiles for pyridoxal and pyridoxal phosphate are very other and to that of lactate dehydrogenase, indicating that both predominantly located to the cytosoI. There are smaff amounts of and pyridoxal phosphate associated with the dense fractions, but no 1.15 g . cmV3 corresponding to alkaline phosphatase.
and pyridoxal neutrophil. The similar to each compounds are both pyridoxaf peak at density
20
1
Free pyridoxal
Lactate dehydrcqenase
Pyrldoxal phosphate
Alkaline
phosphatase
Density
Fig. 1. Isopycnic centrifugation of post-nuciear supernatant prepared from neutrophit leukocyte homogenates. Results show mean distributions from two experiments. Frequency is defined as the fraction of total recovered activity present in the gradient fraction divided by the density span covered. The activity represents enzyme remaining in the sample present over the density span 1.05 g,cm-‘-l.lO’g.cmlayer.
Vmiation in neu~rophi~ p~r~doxa~ and p~ridoxai phosphate
Table I compares the levels of free pyridoxal, and the specific activity of genates prepared from control subjects, and patients with chronic granulocytic
TABLE
pyridoxal, pyridoxal phosphate and total alkaline phosphatase in neutrophil homowomen in the third trimester of pregnancy leukaemia. The variation in alkaline phos-
I
PYRIDOXAL AND PYRIDOXAL PHOSPHATE LEVELS TIVITY IN HUMAN NEUTROPHILIC GRANULOCYTES Controls
Patients in third trimester of pregnancy R.19& 1.57
Pyridoxal Pyridoxal
phosphate
(4) 2X.5 i2.2 36.7
Alkaline
(6) 26.9
phosphatase
(8) **
‘-
7.21 + 1.31 (5) 13.4 +4.0 15)
(6) * Total pyridoxal
AND
i-3.7
f3.2
20.6
iJ.5
(5) 3.23 +0.34 (10)
ALKALINE
PHOSPHATASE
AC‘-
Chronic granulocytic leukaemia patients
9.44 i 0.41 (5) 14.3 * 1.5 (5) 23.7
* 1.9
15) 0.47 + 0. IO (8) **
Results are shown as mean values+ SEM with the number of preparations assayed in duplicate shown between parentheses. Metabolite levels are expressed as pmoI/mg protein and enzyme activites are expressed as mU/mg protein. Significant differences from control subjects analyzed by Student’s f test.
17
phatase activity is again striking, with a 90% reduction in chronic granulocytic leukaemia and a S-fold increase in pregnancy. The levels of pyridoxal phosphate and total pyridoxal are significantly higher in neutrophils from women in late pregnancy when compared to controls, but are unaltered in patients with chronic granulocytic leukaemia. There is very little variation in the level of free pyridoxal between the three groups of subjects. Discussion The present study has shown that human polymorphonuclear leukocytes do contain both pyridoxal and pyridoxal phosphate, which can be reliably measured by both microbiological and fluorimetric methods, confirming previous reports [ 16,171 that leukocytes contain pyridoxal phosphate. The presence of free pyridoxal in these cells has not been previously described. Analytical subcellular fractionation studies have shown that pyridoxal and pyridoxal phosphate are localised almost exclusively to the soluble fraction, with a small amount associated with the high density granules. There was no pyridoxal phosphate associated with the alkaline phosphatase containing granules. In circulating neutrophils pyridoxal phosphate is probably tightly bound as a cofactor to cytosolic enzymes such as aspartate and alanine aminotransferases. The levels of pyridoxal in neutrophils from control subjects and patients who exhibit marked variation in alkaline phosphatase activity were very similar in the three groups. However, there was an increase in the level of pyridoxal phosphate in late pregnancy. These normal or increased levels of leukocyte pyridoxal, and pyridoxal phosphate, respectively, in late pregnancy, do not support the suggestion that pyridoxine deficiency is a frequent finding in pregnancy, a claim based on plasma pyridoxal assays [ 15,181, which suggests that plasma levels may be a poor reflection of tissue deficiency. Increased levels of pyridoxal phosphate is the reverse of what would have been expected if pyridoxal phosphate were directly regulated by the alkaline phosphatase activity, since neutrophils from women in late pregnancy have increased alkaline phosphatase, so a low level of pyridoxal phosphate might have been expected. This study suggests that pyridoxal phosphate is not the principal physiological substrate for human neutrophil alkaline phosphatase, although previous studies [lo] have clearly shown that alkaline phosphatase is capable of hydrolysing pyridoxal phosphate in vitro. Acid pyridoxal phosphatase, which is present in large amounts in these cells [lo] also hydrolyses pyridoxal phosphate and would be expected to play a role in regulating the level of the dephosphorylated vitamer. Attempts to assay pyridoxal kinase with a sensitive radioassay [19] failed to detect this activity in circulating leukocytes. It is therefore likely that pyridoxal phosphorylation occurs in granulocyte precursor cells. The increased amount of pyridoxal phosphate in leukocytes from women in the third trimester of pregnancy remains unexplained.
18
Acknowledgements We are grateful to Mr. H. Gordon and Dr. J. Goldman for providing blood samples from their patients. We would also like to thank Mr. Peter White for expert technical assistance and Ms. Rosamund Greensted for secretarial help. References 1 Spector R, Greenwald L. Transport and metabolism of vitamin $ in rabbit brain and choroid plexus. J Biol Chem 1978; 253: 2373-2379. 2 Merrill AH, Moriike K, McCormack DB. Evidence for the regulation of pyridoxal 5’-phosphate formation in liver by pyridoxamine (pyridoxine) 5’-phosphate oxidase. Biochem Biophys Res Commun 1978; 83: 984-990. 3 Lumeng L, Brashear RE, Li TK. Pyridoxal 5’-phosphate in plasma: source, protein-binding. and cellular transport. J Lab Clin Med 1974; 84: 334-343. 4 Anderson BB, Nemark PA. Rawlins M, Green R. Plasma binding of vitamin $ compounds. Nature 1974; 250: 502-504. 5 Li TK, Lumeng L. Veitch RL. Regulation of pyridoxal 5’-phosphate metabolism in liver. Biochem Biophys Res Commun 1974; 61: 677-684. 6 Okun DB, Tanaka KR. Leukocyte alkaline phosphatase. Am J Haematol 1978; 4: 293-299. 7 Rustin GJS. Peters TJ. Studies on the subcellular organelles of neutrophils in chronic granulocytic leukaemia with special reference to alkaline phosphatase. Br J Haematol 1979; 41: 533-543. 8 Rustin GJS, Wilson PD, Peters TJ. Studies on the subcellular localisation of human neutrophil alkaline phosphatase. J Cell Sci 1979; 36: 401-412. 9 Wilson PD. Rustin GJS, Peters TJ. The ultrastructural localisation of human neutrophil alkaline phosphatase in normal individuals, during pregnancy and in patients with chronic granulocytic leukaemia. Histochem J 1981; 13: 31-43. 10 Smith GP, Peters TJ. Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase. Biochim Biophys Acta 1981; 661: 287-294. 11 Wilson PD. Smith GP. Peters TJ. Pyridoxal 5’-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils. Histochem J 1983, in press. 12 Takanashi S, Matsunaga I, Tamura Z. Fluorometric determination of pyridoxal and its 5’-phosphate in biological materials. J Vitamin01 1970; 16: 129- 131. 13 Segal, AW, Peters TJ. Analytical sub-cellular fractionation of human granulocytes with special reference to the localization of enzymes involved in microbicidal mechanisms. Clin Sci Mol Med 1977: 52: 429-442. 14 Peters TJ. The analytical subcellular fractionation of jejunal biopsy specimens. Methodology and characterisation of the organelles in normal tissue. Clin Sci Mol Med 1976; 5 1: 557-574. 15 Anderson BB, Peart MB, Fulford-Jones CE. The measurement of serum pyridoxal by a microbiological assay using L.acrobacillus casei. J Clin Path 1970; 23: 232-242. 16 Wachstein M, Moore C, Graffeo LW. Pyridoxal phosphate ($-al-PO,) levels of circulating leukocytes in maternal and cord blood. Proc Sot Exp Biol Med 1957; 96: 326-328. 17 Donald EA. Ferguson RF. A micro method for determination of pyridoxal phosphate in leukocytes and liver. Analyt Biochem 1964; 7: 335-341. 18 Wachstein MJD, Kellner JD, Ortiz JM. Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following $ load tests. Proc Sot Exp Biol Med 1960: 103: 350-353. 19 Karawya E, Fonda ML. Purification, assay and kinetic properties of sheep liver pyridoxal kinase. Analyt Biochem 1978; 90: 525-533.
Cfiriica Chintica Acru, 129 (1983) 13-18 Elsevier Biomedical Press
CCA 2439
Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity Gillian
P. Smith
a,*, Barbara
B. Anderson
’ and Timothy
J. Peters
a
u Division oj Clinical Cell Biolop, MRC Clinical Research Centre, Harrow, Middlesex (lJK) und ’ Department
of Haematology,
St. Barthoiemew’s (Received
Hospiral,
West Smithfiefd,
London (UK)
August 24th. 1982)
Summary Neutrophil leukocytes. isolated from normal subjects and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation, showed very similar cytosol distributions of ‘pyridoxal and pyridoxal phosphate and lactate dehydrogenase. The small amounts of pyridoxal and pyridoxal phosphate associated with the dense granule fractions were not associated with the alkaline phosphatase containing granules. The levels of pyridoxal and pyridoxal phosphate were determined in neutrophils from control subjects, women in the third trimester of pregnancy and patients with chronic granulocytic leukaemia. Neutrophil pyridoxal phosphate was increased in women in the third trimester of pregnancy compared to controls, but there was little variation in the level of pyridoxal between the groups. There was no consistent correlation between the pyridoxal phosphate and the neutrophil alkaline phosphatase activity in the patient groups, Although in vitro neutrophil alkaline phosphatase rapidly hydrolyses pyridoxal phosphate, it is suggested that in vivo this is unlikely to be the principal function of the enzyme.
Introduction Pyridoxal 5’-phosphate, the coenzyme form of pyridoxine (vitamin $) has an important function in numerous biochemical reactions. The pyridoxal content of tissues is probably regulated by a number of factors such as plasma membrane
* Correspondence: Watford Road,
0009-898
Dr. G.P. Smith, Division of Clinical Harrow, Middlesex, HA1 3UJ, UK.
I /83/0000-0000/$03.00
0 1983 Else&x
Cell Biology,
Science Publishers
MRC Clinical
Research
Centre.
14
transport [l], by phosphorylation of free pyridoxal [2], and by binding of the coenzyme to protein [3,4]. It has also been shown that cellular phosphatases (EC 3.1.3.-) play an important role in regulating tissue pyridoxal phosphate levels [5]. When the amount of pyridoxal phosphate exceeds the binding capacity of the tissue apoproteins, free pyridoxai phosphate is rapidfy hydrolysed and the released pyridoxal can readily traverse the cell membrane. Normal human polymorphonuclear leukocytes are a rich source of alkaline phosphatase and the levels of activity vary considerably in certain neutrophil disorders and in pregnancy [6,7]. This alkaline phosphatase activity has been localised to a unique organelle (phosphasome) of the neutrophil [8,9], although the physiological function of this organelle is, as yet, unclear. Recently, analytical subcellular fractionation experiments IlO] and EM cytochemical studies [ 1l] have clearly shown that alkaline pyridoxal phosphate phosphatase is also located to the phosphasome granules. In addition to sharing the same subcellular localisation both alkaline pyridoxal phosphate phosphatase and alkaline phosphatase activities shared identical pH optima and Mg”+ requirements, and showed similar inhibition curves with levamisole, a specific inhibitor ,of alkaline phosphatase. Since the specific activities of the enzymes in neutrophils isolated from women in late pregnancy and patients with chronic granulocytic leukaemia varied in a parallel manner when compared to controls, it was suggested that pyridoxal phosphate may be a physiological substrate for neutrophil alkaline phosphatase [IO]. The present study reports the subcellular localisation of pyridoxal and pyridoxal phosphate in human neutrophils using analytical subcellular fractionation techniques and compares the levels of these vitamers in chronic granulocytic leukaemia and pregnancy, conditions known to exhibit striking variations in alkaline phosphatase and alkaline pyridoxal phosphate phosphatase levels compared with control subjects. Materials and methods Preparation of human polymorphon~c~ear leukocytes Polymorphonuclear leukocytes were isolated by dextran sedimentation and Ficoll-Hypaque centrifugation as previously described [7]. Blood, 50 ml, was used directly from control subjects and pregnant women, but blood from leukaemic patients, with a high leukocyte count, was diluted with an equal volume of 0.15 mol/l NaCI. Levels of pyridoxa( and pyridoxal phosphate The neutrophils were resuspended in 4.5 ml ice-cold 0.2 mol/l Na acetate-acetic acid buffer, pH 4.0, and disrupted in a Dounce homogeniser by 30 strokes of a tight-fitting (Type B) pestle. A portion was removed for protein determination and the remainder boiled for 3 min. For the estimation of free pyridoxal 2 ml boiled neutrophils were mixed with 1 ml 0.2 mol/I Na acetate buffer, pH 4.0, and I ml 1.2 mol/I trichloroacetic acid. The mixture was heated at SO’C for 5 min to ensure complete precipitation of the protein. After centrifugation at 800 x g for 5 min the
15
supernatant was removed and retained for analysis. For total pyridoxal 2 ml boiled neutrophils were added ta I ml 0.2 m&,/l Na acetate-acetic acid buffer, pH 4.0, containing 0.34 mU acid phosphatase (potato Type II, Sigma London Ltd. London, UK) and incubated at 37°C for 60 min. This procedure hydrolysed all pyridoxal phosphate present to pyridoxal. After cooling 1 ml 1.2 mol/l trichloroacetic acid was added and the samples treated exactly as those for the estimation of free pyridoxal. The level of pyridoxal in the extracts was estimated with a modification of the fluorimetric method of Takanashi et al ]12].
Subeeklar
locaiisation ofpyridoxal and pyridoxal phosphate
The isolated neutrophils were pelleted in 4.5 ml 0.2 mol/l sucrose medium containing 1 mmol/l Na, EDTA, pH 7.2, and 5000 units heparin/I, and disrupted in a Dounce homogeniser as described previously [13]. The homogenate was centrifuged at 800 x g for 10 min and the post-nuclear su~ernatant removed, stored on ice and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation [14]. ApproximateIy 5 ml of the postnuclear supernatant was Iayered onto a 28 ml sucrose density gradient, extending linearly with respect to volume, from a density of 1.05 g * crK’ to one of 1.28 g. cmV3 and resting on a 6 ml cushion of density 1.32 g - cm -’ in a Beaufay automatic zonal rotor. All gradient solutions contained 1 mmol/t Na, EDTA pH 7.2, and 5000 units heparin/l and were prepared in double distilled deionised water which had been passed through a Millipore filter. The rotor was run at 35000 rev/min for 3.5 min and, after slowing to 8000 rev/min, some 15 fractions were collected, weighed and their density determined as described previously (141. Part of each fraction and the post-nuclear supernatant was retained for enzymic analysis, and the remainder heated in a boihng water bath for 5 min. One ml of each fraction was then treated and extracted in the same way as the cell homogenates, prior to estimation of free and total pyridoxal. After centrifugation the supernatants were extracted with 3 x 8 ml of ether and the final extract made up to a suitable volume with deionised water. The Level of pyridoxal was determined by the microbiological method of Anderson et al ]15], using ~ctobar~~~us casei as assay organism. The difference between the total and free pyridoxal represented pyridoxal phosphate. Corrections were made for the trace levels of pyridoxal and pyridoxai phosphate contents of the sucrose density gradient solutions. Patient groups studied were as reported previously [7]. Results
~~bcet~ular ~oca~~~ation of ~~r~doxai and ~~rjdoxa~ phosphate Fig. 1 shows the mean density gradient distribution of pyridoxal phosphate and some of the organelle marker enzymes of the human distribution profiles for pyridoxal and pyridoxal phosphate are very other and to that of lactate dehydrogenase, indicating that both predominantly located to the cytosoI. There are smaff amounts of and pyridoxal phosphate associated with the dense fractions, but no 1.15 g . cmV3 corresponding to alkaline phosphatase.
and pyridoxal neutrophil. The similar to each compounds are both pyridoxaf peak at density
20
1
Free pyridoxal
Lactate dehydrcqenase
Pyrldoxal phosphate
Alkaline
phosphatase
Density
Fig. 1. Isopycnic centrifugation of post-nuciear supernatant prepared from neutrophit leukocyte homogenates. Results show mean distributions from two experiments. Frequency is defined as the fraction of total recovered activity present in the gradient fraction divided by the density span covered. The activity represents enzyme remaining in the sample present over the density span 1.05 g,cm-‘-l.lO’g.cmlayer.
Vmiation in neu~rophi~ p~r~doxa~ and p~ridoxai phosphate
Table I compares the levels of free pyridoxal, and the specific activity of genates prepared from control subjects, and patients with chronic granulocytic
TABLE
pyridoxal, pyridoxal phosphate and total alkaline phosphatase in neutrophil homowomen in the third trimester of pregnancy leukaemia. The variation in alkaline phos-
I
PYRIDOXAL AND PYRIDOXAL PHOSPHATE LEVELS TIVITY IN HUMAN NEUTROPHILIC GRANULOCYTES Controls
Patients in third trimester of pregnancy R.19& 1.57
Pyridoxal Pyridoxal
phosphate
(4) 2X.5 i2.2 36.7
Alkaline
(6) 26.9
phosphatase
(8) **
‘-
7.21 + 1.31 (5) 13.4 +4.0 15)
(6) * Total pyridoxal
AND
i-3.7
f3.2
20.6
iJ.5
(5) 3.23 +0.34 (10)
ALKALINE
PHOSPHATASE
AC‘-
Chronic granulocytic leukaemia patients
9.44 i 0.41 (5) 14.3 * 1.5 (5) 23.7
* 1.9
15) 0.47 + 0. IO (8) **
Results are shown as mean values+ SEM with the number of preparations assayed in duplicate shown between parentheses. Metabolite levels are expressed as pmoI/mg protein and enzyme activites are expressed as mU/mg protein. Significant differences from control subjects analyzed by Student’s f test.
17
phatase activity is again striking, with a 90% reduction in chronic granulocytic leukaemia and a S-fold increase in pregnancy. The levels of pyridoxal phosphate and total pyridoxal are significantly higher in neutrophils from women in late pregnancy when compared to controls, but are unaltered in patients with chronic granulocytic leukaemia. There is very little variation in the level of free pyridoxal between the three groups of subjects. Discussion The present study has shown that human polymorphonuclear leukocytes do contain both pyridoxal and pyridoxal phosphate, which can be reliably measured by both microbiological and fluorimetric methods, confirming previous reports [ 16,171 that leukocytes contain pyridoxal phosphate. The presence of free pyridoxal in these cells has not been previously described. Analytical subcellular fractionation studies have shown that pyridoxal and pyridoxal phosphate are localised almost exclusively to the soluble fraction, with a small amount associated with the high density granules. There was no pyridoxal phosphate associated with the alkaline phosphatase containing granules. In circulating neutrophils pyridoxal phosphate is probably tightly bound as a cofactor to cytosolic enzymes such as aspartate and alanine aminotransferases. The levels of pyridoxal in neutrophils from control subjects and patients who exhibit marked variation in alkaline phosphatase activity were very similar in the three groups. However, there was an increase in the level of pyridoxal phosphate in late pregnancy. These normal or increased levels of leukocyte pyridoxal, and pyridoxal phosphate, respectively, in late pregnancy, do not support the suggestion that pyridoxine deficiency is a frequent finding in pregnancy, a claim based on plasma pyridoxal assays [ 15,181, which suggests that plasma levels may be a poor reflection of tissue deficiency. Increased levels of pyridoxal phosphate is the reverse of what would have been expected if pyridoxal phosphate were directly regulated by the alkaline phosphatase activity, since neutrophils from women in late pregnancy have increased alkaline phosphatase, so a low level of pyridoxal phosphate might have been expected. This study suggests that pyridoxal phosphate is not the principal physiological substrate for human neutrophil alkaline phosphatase, although previous studies [lo] have clearly shown that alkaline phosphatase is capable of hydrolysing pyridoxal phosphate in vitro. Acid pyridoxal phosphatase, which is present in large amounts in these cells [lo] also hydrolyses pyridoxal phosphate and would be expected to play a role in regulating the level of the dephosphorylated vitamer. Attempts to assay pyridoxal kinase with a sensitive radioassay [19] failed to detect this activity in circulating leukocytes. It is therefore likely that pyridoxal phosphorylation occurs in granulocyte precursor cells. The increased amount of pyridoxal phosphate in leukocytes from women in the third trimester of pregnancy remains unexplained.
18
Acknowledgements We are grateful to Mr. H. Gordon and Dr. J. Goldman for providing blood samples from their patients. We would also like to thank Mr. Peter White for expert technical assistance and Ms. Rosamund Greensted for secretarial help. References 1 Spector R, Greenwald L. Transport and metabolism of vitamin $ in rabbit brain and choroid plexus. J Biol Chem 1978; 253: 2373-2379. 2 Merrill AH, Moriike K, McCormack DB. Evidence for the regulation of pyridoxal 5’-phosphate formation in liver by pyridoxamine (pyridoxine) 5’-phosphate oxidase. Biochem Biophys Res Commun 1978; 83: 984-990. 3 Lumeng L, Brashear RE, Li TK. Pyridoxal 5’-phosphate in plasma: source, protein-binding. and cellular transport. J Lab Clin Med 1974; 84: 334-343. 4 Anderson BB, Nemark PA. Rawlins M, Green R. Plasma binding of vitamin $ compounds. Nature 1974; 250: 502-504. 5 Li TK, Lumeng L. Veitch RL. Regulation of pyridoxal 5’-phosphate metabolism in liver. Biochem Biophys Res Commun 1974; 61: 677-684. 6 Okun DB, Tanaka KR. Leukocyte alkaline phosphatase. Am J Haematol 1978; 4: 293-299. 7 Rustin GJS. Peters TJ. Studies on the subcellular organelles of neutrophils in chronic granulocytic leukaemia with special reference to alkaline phosphatase. Br J Haematol 1979; 41: 533-543. 8 Rustin GJS, Wilson PD, Peters TJ. Studies on the subcellular localisation of human neutrophil alkaline phosphatase. J Cell Sci 1979; 36: 401-412. 9 Wilson PD. Rustin GJS, Peters TJ. The ultrastructural localisation of human neutrophil alkaline phosphatase in normal individuals, during pregnancy and in patients with chronic granulocytic leukaemia. Histochem J 1981; 13: 31-43. 10 Smith GP, Peters TJ. Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase. Biochim Biophys Acta 1981; 661: 287-294. 11 Wilson PD. Smith GP. Peters TJ. Pyridoxal 5’-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils. Histochem J 1983, in press. 12 Takanashi S, Matsunaga I, Tamura Z. Fluorometric determination of pyridoxal and its 5’-phosphate in biological materials. J Vitamin01 1970; 16: 129- 131. 13 Segal, AW, Peters TJ. Analytical sub-cellular fractionation of human granulocytes with special reference to the localization of enzymes involved in microbicidal mechanisms. Clin Sci Mol Med 1977: 52: 429-442. 14 Peters TJ. The analytical subcellular fractionation of jejunal biopsy specimens. Methodology and characterisation of the organelles in normal tissue. Clin Sci Mol Med 1976; 5 1: 557-574. 15 Anderson BB, Peart MB, Fulford-Jones CE. The measurement of serum pyridoxal by a microbiological assay using L.acrobacillus casei. J Clin Path 1970; 23: 232-242. 16 Wachstein M, Moore C, Graffeo LW. Pyridoxal phosphate ($-al-PO,) levels of circulating leukocytes in maternal and cord blood. Proc Sot Exp Biol Med 1957; 96: 326-328. 17 Donald EA. Ferguson RF. A micro method for determination of pyridoxal phosphate in leukocytes and liver. Analyt Biochem 1964; 7: 335-341. 18 Wachstein MJD, Kellner JD, Ortiz JM. Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following $ load tests. Proc Sot Exp Biol Med 1960: 103: 350-353. 19 Karawya E, Fonda ML. Purification, assay and kinetic properties of sheep liver pyridoxal kinase. Analyt Biochem 1978; 90: 525-533.