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Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Accumulation of Erythrocyte Nucleotides and Their Pattern in Lead Workers a
a
Tadashi Sakai Ph.D. , Takaharu Araki M.D. & Koichi Ushio M.D.
a
a
Center of Occupational Medicine , Tokyo Labor Accident Hospital , Tokyo, Japan Published online: 03 Aug 2010.
To cite this article: Tadashi Sakai Ph.D. , Takaharu Araki M.D. & Koichi Ushio M.D. (1990) Accumulation of Erythrocyte Nucleotides and Their Pattern in Lead Workers, Archives of Environmental Health: An International Journal, 45:5, 273-277, DOI: 10.1080/00039896.1990.10118745 To link to this article: http://dx.doi.org/10.1080/00039896.1990.10118745
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Accumulation of Erythrocyte Nucleotides and Their Pattern in
Downloaded by [University of Glasgow] at 01:31 20 December 2014
Lead Workers
TADASHI SAKAI, Ph.D. TAKAHARU ARAKI, M.D. KOlCHl USHIO, M.D. Center of Occupational Medicine Tokyo labor Accident Hospital Tokyo, Japan
ABSTRACT. Nucleotides in erythrocytes of lead-exposed subjects were analyzed by highperformance liquid chromatography (HPLC). Most of the pyrimidine levels correlated well with blood lead concentrations (Pb-6) and pyrimidine 5’-nucleotidase (P5N) activity. Highly significant correlations were found between Pb-B and uridine 5’-diphosphate-glucose (UDPC), cytidine 5’-triphosphate (CTP), or CDP-choline (CDPC). The levels of these compounds were sharply elevated when P5N activity was reduced to levels less than 7pmole/ hag hemoglobin (Hb), which corresponded to a Pb-B of 60 pel100 g. Therefore, concentration of these nucleotides may provide a useful index of lead poisoning. Adenosine 5’triphospate (ATP) concentrations were correlated negatively with Pb-6, whereas adenosine 5‘-monophosphate (AMP) concentrations were correlated positively with Pb-6. These results suggest that lead affects not only pyrimidine nucleotide metabolism but also purine nucleotide metabolism (energy production system).
IN LEAD WORKERS, erythrocyte enzyme pyrimidine 5’-nucleotidase (P5N) activity declines linearly with increasing blood lead concentrations (Pb-B) between 10 and 100 kg/lOO g, and it can be used for an indicator of lead exposure.’ Congenital deficiency of this enzyme results in nonspherocytic hemolytic anemia in which the erythrocytes contain large amounts of pyrimidine nucleotides and show pronounced basophilic stippling (BSE).* The accumulated pyrimidine compounds appear to cause feedback inhibition of ribonucleic acid (RNA) catabol i s m ~ Undegraded .~ ribosomes aggregate to produce BSE. Accumulation of pyrimidine compounds in erythrocytes also affects glucose-6-phosphatedehydrogenase and suppresses pentose phosphate cycle activity, which results in hem~lysis.~ The mechanism of hemolysis in lead poisoning appears similar to SepternberlOctober 1990 [Vol. 45 (No. 5)]
that postulated in hereditary P5N deficiency (P5ND). However, the composition of the nucleotides that accumulated in erythrocytes of lead workers and the lead effect on the nucleotide pool (other than pyrimidine) have not been reported. In the present study, we report on the development of a method for determining erythrocyte nucleotides using highperformance liquid chromatography (HPLC), and we examine the metabolic disturbances of red cell nucleotide pool by lead exposure. Materials and methods Heparinized venous blood was obtained from 47 lead workers in secondary smelters and from 40 lead workers in printing offices. Nucleotides in erythrocytes were extracted from 0.5 ml of fresh blood with 273
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1 ml of 0.6N perchloric acid. After centrifigation at 3 000 rpm for 10 min, the supernatant was neutralized with an equal volume of 0.2M K,CO,, and 100 FI of the solution was used for the HPLC analysis. The HPLC apparatus from Waters Assoc, (Milford, MA, USA) consisted of two pumps (Model 510); an automatic sample injector (WISP 7108); an automated gradient controller; a temperature control system; and a programmable multiwavelength detector (Model 490) and a chromatogram proccessor (Model 70008) from System Instruments Corporation (Tokyo, Japan)were used. Column temperature and detector wavelength were set at 70 “C and 270 nm, respectively. The column (4.6 x 150 mm) used was packed with an anion exchanger, Hitachi Gel #3013 N (Hitachi, Tokyo, Japan). The eluents for HPLC were solution A (20 mM KH,PO,, 40 mM CH,COONH,, 40 m M NH,CI, 6% CH,CN) and solution B (50 m M K,HPO,, 50 m M KH,PO,, 300 mM NH,CI, 6% CH,CN). After an isocratic period of 10 min with solution A, we applied a linear gradient from 0 to 100% of solution B during the next 50 min, then kept the eluent at 100% of solution B for 10 min. The flow rate was 0.5 ml/min during the separation of nucleotides, and the mobile phase was then restored to 100% solvent A at a flow rate of 1.0 ml/min. After re-equilibration with solution A for 20 min, the flow rate was decreased to 0.5 m l h i n , and a new sample was injected. Reference standards used for the identification of nucleotide peaks were obtained from Seikagaku Kogyo Co., LTD (Tokyo, Japan).The P5N activity, Pb-B, and zinc protoporphyrin (ZP) were also determined using the same blood samples by the methods reported previo~sly.’,~
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Figure 1 shows typical chromatographic separations of nucleotides in erythrocytes. In the present HPLC system, 14 species of nucleotides were separated from each other and identified by cochromatography with reference standards. In a control subject (Fig. l a ) , the major nucleotides found in erythrocytes were purine nucleotides (i.e., tri- and di-phosphate). Erythrocytes from a lead worker (Fig. I b ) showed significant increases in levels of pyrimidine nucleotides, which were absent or found only at low levels in control subjects. In a lead worker, the major elevated peaks are cytidine 5’-triphosphate (CTP), uridine 5’-diphosphate-glucose (UDPG), CDP-choline (CDPC), and diphosphate of pyrimidine nucleosides. In contrast, monophosphate of pyrimidine nucleoside, which is the direct substrate for P5N, was present in trace amounts-even in erythrocytes from a lead worker. Thus, the prominent pyrimidine nucleotides in erythrocytes from lead workers were in the form of triphosphates or of complex compounds with sugar or choline, which are rich in energy rather than monophosphates. The nucleotide profiles in erythrocytes were changed during storage of blood at 4 “C (Fig. I c ) . Compared to fresh samples, the triphosphates of purine and 274
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Fig. 1.
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40 60 Retention Time (min)
HPLC separation of 14 nucleotides (A-N) in blood extracts.
Blood was obtained from a control subject (a) and a lead worker (b and c). Blood was extracted 30 min (a and b) and 24 h ( c ) after sampling. (A = C D K , B = CMP, C = UMP, D = AMP, E = CMP, F = UDPC, C=CDP, H=UDP, I=ADP, J=CDP, K=CTP, L=UTP, M=ATP, and N = CTP). Archives of Environmental Health
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pyrimidine nucleosides diminished, and mono- and diphosphates increased, indicating the rapid degradation of triphosphate to form mono- and diphosphates of nucleosides. Half-lives of decay of nucleotides during storage at 4 "C were calculated by the equation for the decay curves, some of which are shown in Fig. 2. Adenosine 5'-triphosphate (ATP) showed the highest rate of degradation, and CDPC and UDPG showed the slower rate. Half-lives of ATP, CTP, GTP, uridine 5'-triphosphate (UTP), CDPC, and UDPG were 11.0,14.3,16.5, 38.2,170, and 376 h, respectively, in a lead worker whose Pb-B level was 42.8 pg/lOO g. In a control subject, the half-lives of ATP, GTP, and UDPG were 10.7, 13.7, and 150 h, respectively. No difference in the decay curves were found between the two subjects. Recovery of the 14 species of spiked nucleotides ranged between 84 and 97%, and the coefficient of variation (cv)for the nucleotide analyses was between 3.8 and 8.9% ( n = 10). We next adopted the present method to blood obtained from 47 lead workers. Extraction of nucleotides was conducted 3-4 h after sampling. Means f standard deviations (ranges) of Pb-B and P5N were 42.2 & 21.9 (9.9-112) p.g/lOO g and 10.2 f 3.7 (3.219.4) pmol/h . g hemoglobin (Hb), respectively. The P5N activity declined with increasing P b B concentrations and corrilated well with Pb-B (Fig. 3A). Table 1 shows the erythrocyte nucleotide concentrations and their correlations vs. Pb-B or P5N activity. Many kinds of nucleotides examined were correlated significantly with Pb-B and P5N activity. Highest correlations were found in Pb-B vs. UDPG, CTP, or CDPC (Fig. 4A, B, and C). These nucleotide levels were also negatively correlated with P5N activity (Fig. 5A, B, and C). Although the correlations improved when the values of P5N activity were transformed to logarithmic scale (Table I ) , the nucleotides were still scattered in a two-phase distribution (fig. 5A and B). This pattern of distribution results from the fact that the pyrimidine nucleotide levels were sharply elevated as P5N activity was suppressed to levels less
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Fig. 3. Relationship between Pb-B and P5N activity or purine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted)close to regressionlines show 95% confidence ranges of the regression line, and outmost curves (dotted) indicate 95% predictive intervals of individual values. (A) = P5N log y = 0.00726~ + 1.28, (B)=ATP log y = 0.00245~ 4.89, and (C)=AMP y =
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Fig. 2. Decay curves for nucleotidedegradation in blood from a lead worker during storage at 4OC after sampling. O = U D P C (log y= -0.000758~ 2), O=CDPC (log y = -0.00170~ 2), .=CTP (log y = 0.021lx 2),and O=ATP (log y = -0.0282~ 2).
-
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September/Odober 1990 [Vol. 45 (No. 5)]
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than 7 pmole/h-g Hb, which corresponds to a Pb-B concentration of 60 pg/lOO g (Fig. 3A). The elevated levels (mean standard deviation) of UDPG, CTP, and CDPC were 4 096 1 252,l 062 f 508, and 928 ? 319 nmol/dl, respectively, in 6 workers who Pb-6 levels exceeded 60 pg/lOO g. The mean concentrations ? standard deviations of UDPG, CTP, and CDPC in lead workers (n = 40)whose Pb-B were less than 20 pg/lOOg were 2 200 888,266 362, and 472 247 nmol/dl, respectively. Concentrations of ATP correlate negatively with PbB, whereas AMP concen-
*
*
*
275
tration correlates positively (Fig. 38 and C). The correlation of ATP vs. Pb-B, both of which were corrected by Hct, was also siginificant (data not shown). In 47 lead workers, energy charge (y) was calculated from the equation, (ADPI2
+ ATP)/(AMP + ADP + ATP)
X
100 (Yo),
and was significantly decreased as Pb-B increased ( x ) (y = - 0 . 0 6 6 ~ 93.7, r = 0.557). These results suggest that lead affects pyrimidine nucleotide metabolism and purine nucleotide metabolism (energy production system). The ATP levels of 40 lead workers whose Pb-B were less than 20 bg/lOOg were 64 600 2
+
8 573 nmol/dl (nucleotides were extracted 4 h after sampling of blood). Discussion
Using anion-exchange HPLC to study lead-treated animals, Angle et a1.6 found three nucleotides (i.e., UTP, CTP, and CDP) that were increased in blood cells. In the present study, 14 species of nucleotides were separated, including CDPC and UDPG, which are the major nucleotides that are correlated with depression of P5N activity in lead-exposed subjects. This i s the first study that reports the composition of
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Fig. 4. Relation between Pb-B and pyrimidine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted) close to regression lines show 95% confidence ranges of the regression line, and outmost curves (doffed) indicate 95% predictive intervals of individual values. (A) = UDPC y = 3 3 . 7 ~ 782, (B) = CTP y = 1 3 . 6 ~- 307,and (C)=CDPC y = 8 . 3 8 ~ 119.
+
300
125
Fig. 5. Relationship between P5N activity and pyrimdine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted)close to regression lines show 95% confidence ranges of the regression line, and outmost curves (dotted) indicate 95% predictive intervals of individual values. (A) = UDPC log y = 0.0001 55x 1.32, (B) = CTP log y = - 0.000395~ 1.08, and (C) = CDPC 1- y = - 0.000492~ 1.21.
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Archives of Environmental Health
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Table 1.-Erythrocyte Nucleotide Concentrations in 47 Lead Workers and Their Correlations vs. Blood lead Concentration (Pb-B) or Pyrimidine 5-Nucleotidase (P5N) Activity
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Concentration (nmol/dl blood) Nucleotides
Mean
CDPC CMP UMP AMP GMP UDPC CDP UDP ADP GDP CTP UTP ATP CTP
472 19 51 1640 109 2 210 33 208 10 020 1012 266 956 62 600 2 981
SO 246 36 43 935 103
888 36 187 1 850 169 362 286 10 400 386
Correlation coefficients Pb-B VS. nucleotides 0.744 0.278 0.618 0.442 0.233 0.831
0.718 0.307 0.467 0.337 0.270
0.484
0.280 0.533 - 0.214 0.017 0.676 0.301 - 0.687 -0.393
0.617 - 0.191 0.016 0.823
0.406 -0.640 -0.369
abnormal nucleotides that accumulated in red blood cells of workers who were occupationally exposed to lead. The pyrimidine diphosphodiesters, CDPC and UDPG, and CTP are the most prominent abnormal nucleotides in blood cells of lead-exposed subjects. The nucleotides that were increased in lead workers are similar to those found in P5ND,7,8,9,but the concentrations are much lower than the massive amounts found in PSND. In the present study, we found that the levels of purine nucleotides (other than pyrimidine nucleotides) were also affected by lead exposure, suggesting disturbances of the energy production system by lead. As Pb-B values rose, ATP levels showed a slight reduction, whereas AMP levels were correlated positively with Pb-B (Fig. 3B and C). Energy charges were consequently decreased in workers who had Pb-B greater than 60 p.g/lOO g. Angle et a1,6,10 however, found that purine nucleotides were unchanged in red cells from lead-poisoned rabbits with mean Pb-B levels of 69.4 p.g/dl and in children with Pb-B levels between 30 and 72 p.g/dl. The energy charge was also normal in two children with P5ND.9 The observed pattern of glycolytic intermediates in lead poisoning also suggested that the activity of the EmbdenMeyerhof pathway was However, Torrance and Whittaker’ found that the concentrations of ADP and AMP in red blood cells of PSND were between 2 and 3 times normal, and the distribution of adenine nucleotides was abnormal with the lower energy forms being favored.
* * * * * * * * * * Submitted for publication August 7, 1989; revised; accepted for publication March 14,1990. Requests for reprints should be sent to: Tadashi Sakai, Ph.D., Center of Occupational Medicine, Tokyo Labor Accident Hospital, 13-21,Omoriminami-4, Ota-Ku, Tokyo 143, Japan.
!jeptember/October 1990 [Vol. 45 (No. 5)]
Pb-B VS. log nucleotides
0.800
P5N vs. nucleotides
log PSN vs. nucleotides
-0.591 0.051
-0.662 - 0.035 -0.556
-0.289 -0.064 - 0.603
- 0.345
- 0.448 - 0.444 - 0.395 0.227 0.232 - 0.638 -0.274
0.600 0.456
-0.051 -0.749 - 0.4% -0.439 0.256 0.208 -0.779 - 0.317 0.671 0.473
* * * * * * * * * * References 1. Sakai T, Araki T, Ushio K. Determination of pyrimidine 5’nucleotidase (P5N) activity in whole blood as an index of lead exposure. Br J Ind Med 1988;45:420-25. 2. Valentine WN, Fink K, Paglia DE, Harris SR, Adams WS. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5‘nucleotidase deficiency. J Clin Invest 1974;54:866-79. 3. Valentine WN, Tanaka KR, Paglia DE. Hemolytic anemia and erythrocyte enzymopathies. An Int Med 1985;103:245-57. 4. Tomoda A, Noble NA, Lachant NA, Tanaka KR. Hemolytic anemia in hereditary pyrimidine 5’-nucleotidase deficiency: nucleotide inhibition of G6PD and the pentose phosphate shunt. Blood 1982;60:1212-18. 5. Sakai T, Ushio K. A simplified method for determining erythrocyte pyrimidine 5’-nucleotidase (P5N) activity by HPLC and its value in monitoring lead exposure. Br J Ind Med 1986; 43 :839-44. 6. Angle CR, Stohs SJ, Mclntire MS, Swanson MS, Rovang KS. Lead-induced accumulation of erythrocyte pyrimidine nucleotides in rabbit. Toxicol Appl Phamacol 1980;54:161-67. 7. Torrance ID, Whittaker D. Distribution of erythrocyte nucleotides in pyrimidine 5’-nucleotidase deficiency. Br J Haematol 1979;43 ~423-34. 8. Swanson MS, Markin RS, Stohs SJ, Angle CR. Identification of cytidine diphosphodiesterase in erythrocytes from a patient with pyrimidine nucleotidase deficiency. Blood 1984;63:66570. 9. Ericson A, de Verdier CH, Hansen THR, Seip M. Erythrocyte nucleotide pattern in two children in a Norwegian family with pyrimidine S‘-nucleotidase deficiency. Clin Chim Acta 1983; 134%-33. 10. Angle CR, Mclntire MS, Swanson MS, Stohs SJ. Erythrocyte nucleotides in children-increased blood lead and cytidine triohomhate. Pediatr Res 1982:16:331-34. 11. M/wa lshida Y, Takegawa S; Urata G , Toyoda T. A case of lead intoxication: clinical and biochemical studies. Am J Hematoll981 ;1199-105. 12. Valentine WN, Paglia DE, Fink K. Lead poisoning. Association with hemolytic anemia, basophilic stippling, erythrocyte pyrimidine 5’-nucleotidase deficiency, and intraerythrocytic accumulation of pyrimidines. J Clin Invest 1976;58:926-32.
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Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Accumulation of Erythrocyte Nucleotides and Their Pattern in Lead Workers a
a
Tadashi Sakai Ph.D. , Takaharu Araki M.D. & Koichi Ushio M.D.
a
a
Center of Occupational Medicine , Tokyo Labor Accident Hospital , Tokyo, Japan Published online: 03 Aug 2010.
To cite this article: Tadashi Sakai Ph.D. , Takaharu Araki M.D. & Koichi Ushio M.D. (1990) Accumulation of Erythrocyte Nucleotides and Their Pattern in Lead Workers, Archives of Environmental Health: An International Journal, 45:5, 273-277, DOI: 10.1080/00039896.1990.10118745 To link to this article: http://dx.doi.org/10.1080/00039896.1990.10118745
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Accumulation of Erythrocyte Nucleotides and Their Pattern in
Downloaded by [University of Glasgow] at 01:31 20 December 2014
Lead Workers
TADASHI SAKAI, Ph.D. TAKAHARU ARAKI, M.D. KOlCHl USHIO, M.D. Center of Occupational Medicine Tokyo labor Accident Hospital Tokyo, Japan
ABSTRACT. Nucleotides in erythrocytes of lead-exposed subjects were analyzed by highperformance liquid chromatography (HPLC). Most of the pyrimidine levels correlated well with blood lead concentrations (Pb-6) and pyrimidine 5’-nucleotidase (P5N) activity. Highly significant correlations were found between Pb-B and uridine 5’-diphosphate-glucose (UDPC), cytidine 5’-triphosphate (CTP), or CDP-choline (CDPC). The levels of these compounds were sharply elevated when P5N activity was reduced to levels less than 7pmole/ hag hemoglobin (Hb), which corresponded to a Pb-B of 60 pel100 g. Therefore, concentration of these nucleotides may provide a useful index of lead poisoning. Adenosine 5’triphospate (ATP) concentrations were correlated negatively with Pb-6, whereas adenosine 5‘-monophosphate (AMP) concentrations were correlated positively with Pb-6. These results suggest that lead affects not only pyrimidine nucleotide metabolism but also purine nucleotide metabolism (energy production system).
IN LEAD WORKERS, erythrocyte enzyme pyrimidine 5’-nucleotidase (P5N) activity declines linearly with increasing blood lead concentrations (Pb-B) between 10 and 100 kg/lOO g, and it can be used for an indicator of lead exposure.’ Congenital deficiency of this enzyme results in nonspherocytic hemolytic anemia in which the erythrocytes contain large amounts of pyrimidine nucleotides and show pronounced basophilic stippling (BSE).* The accumulated pyrimidine compounds appear to cause feedback inhibition of ribonucleic acid (RNA) catabol i s m ~ Undegraded .~ ribosomes aggregate to produce BSE. Accumulation of pyrimidine compounds in erythrocytes also affects glucose-6-phosphatedehydrogenase and suppresses pentose phosphate cycle activity, which results in hem~lysis.~ The mechanism of hemolysis in lead poisoning appears similar to SepternberlOctober 1990 [Vol. 45 (No. 5)]
that postulated in hereditary P5N deficiency (P5ND). However, the composition of the nucleotides that accumulated in erythrocytes of lead workers and the lead effect on the nucleotide pool (other than pyrimidine) have not been reported. In the present study, we report on the development of a method for determining erythrocyte nucleotides using highperformance liquid chromatography (HPLC), and we examine the metabolic disturbances of red cell nucleotide pool by lead exposure. Materials and methods Heparinized venous blood was obtained from 47 lead workers in secondary smelters and from 40 lead workers in printing offices. Nucleotides in erythrocytes were extracted from 0.5 ml of fresh blood with 273
Downloaded by [University of Glasgow] at 01:31 20 December 2014
1 ml of 0.6N perchloric acid. After centrifigation at 3 000 rpm for 10 min, the supernatant was neutralized with an equal volume of 0.2M K,CO,, and 100 FI of the solution was used for the HPLC analysis. The HPLC apparatus from Waters Assoc, (Milford, MA, USA) consisted of two pumps (Model 510); an automatic sample injector (WISP 7108); an automated gradient controller; a temperature control system; and a programmable multiwavelength detector (Model 490) and a chromatogram proccessor (Model 70008) from System Instruments Corporation (Tokyo, Japan)were used. Column temperature and detector wavelength were set at 70 “C and 270 nm, respectively. The column (4.6 x 150 mm) used was packed with an anion exchanger, Hitachi Gel #3013 N (Hitachi, Tokyo, Japan). The eluents for HPLC were solution A (20 mM KH,PO,, 40 mM CH,COONH,, 40 m M NH,CI, 6% CH,CN) and solution B (50 m M K,HPO,, 50 m M KH,PO,, 300 mM NH,CI, 6% CH,CN). After an isocratic period of 10 min with solution A, we applied a linear gradient from 0 to 100% of solution B during the next 50 min, then kept the eluent at 100% of solution B for 10 min. The flow rate was 0.5 ml/min during the separation of nucleotides, and the mobile phase was then restored to 100% solvent A at a flow rate of 1.0 ml/min. After re-equilibration with solution A for 20 min, the flow rate was decreased to 0.5 m l h i n , and a new sample was injected. Reference standards used for the identification of nucleotide peaks were obtained from Seikagaku Kogyo Co., LTD (Tokyo, Japan).The P5N activity, Pb-B, and zinc protoporphyrin (ZP) were also determined using the same blood samples by the methods reported previo~sly.’,~
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Figure 1 shows typical chromatographic separations of nucleotides in erythrocytes. In the present HPLC system, 14 species of nucleotides were separated from each other and identified by cochromatography with reference standards. In a control subject (Fig. l a ) , the major nucleotides found in erythrocytes were purine nucleotides (i.e., tri- and di-phosphate). Erythrocytes from a lead worker (Fig. I b ) showed significant increases in levels of pyrimidine nucleotides, which were absent or found only at low levels in control subjects. In a lead worker, the major elevated peaks are cytidine 5’-triphosphate (CTP), uridine 5’-diphosphate-glucose (UDPG), CDP-choline (CDPC), and diphosphate of pyrimidine nucleosides. In contrast, monophosphate of pyrimidine nucleoside, which is the direct substrate for P5N, was present in trace amounts-even in erythrocytes from a lead worker. Thus, the prominent pyrimidine nucleotides in erythrocytes from lead workers were in the form of triphosphates or of complex compounds with sugar or choline, which are rich in energy rather than monophosphates. The nucleotide profiles in erythrocytes were changed during storage of blood at 4 “C (Fig. I c ) . Compared to fresh samples, the triphosphates of purine and 274
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Fig. 1.
I
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HPLC separation of 14 nucleotides (A-N) in blood extracts.
Blood was obtained from a control subject (a) and a lead worker (b and c). Blood was extracted 30 min (a and b) and 24 h ( c ) after sampling. (A = C D K , B = CMP, C = UMP, D = AMP, E = CMP, F = UDPC, C=CDP, H=UDP, I=ADP, J=CDP, K=CTP, L=UTP, M=ATP, and N = CTP). Archives of Environmental Health
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pyrimidine nucleosides diminished, and mono- and diphosphates increased, indicating the rapid degradation of triphosphate to form mono- and diphosphates of nucleosides. Half-lives of decay of nucleotides during storage at 4 "C were calculated by the equation for the decay curves, some of which are shown in Fig. 2. Adenosine 5'-triphosphate (ATP) showed the highest rate of degradation, and CDPC and UDPG showed the slower rate. Half-lives of ATP, CTP, GTP, uridine 5'-triphosphate (UTP), CDPC, and UDPG were 11.0,14.3,16.5, 38.2,170, and 376 h, respectively, in a lead worker whose Pb-B level was 42.8 pg/lOO g. In a control subject, the half-lives of ATP, GTP, and UDPG were 10.7, 13.7, and 150 h, respectively. No difference in the decay curves were found between the two subjects. Recovery of the 14 species of spiked nucleotides ranged between 84 and 97%, and the coefficient of variation (cv)for the nucleotide analyses was between 3.8 and 8.9% ( n = 10). We next adopted the present method to blood obtained from 47 lead workers. Extraction of nucleotides was conducted 3-4 h after sampling. Means f standard deviations (ranges) of Pb-B and P5N were 42.2 & 21.9 (9.9-112) p.g/lOO g and 10.2 f 3.7 (3.219.4) pmol/h . g hemoglobin (Hb), respectively. The P5N activity declined with increasing P b B concentrations and corrilated well with Pb-B (Fig. 3A). Table 1 shows the erythrocyte nucleotide concentrations and their correlations vs. Pb-B or P5N activity. Many kinds of nucleotides examined were correlated significantly with Pb-B and P5N activity. Highest correlations were found in Pb-B vs. UDPG, CTP, or CDPC (Fig. 4A, B, and C). These nucleotide levels were also negatively correlated with P5N activity (Fig. 5A, B, and C). Although the correlations improved when the values of P5N activity were transformed to logarithmic scale (Table I ) , the nucleotides were still scattered in a two-phase distribution (fig. 5A and B). This pattern of distribution results from the fact that the pyrimidine nucleotide levels were sharply elevated as P5N activity was suppressed to levels less
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Fig. 3. Relationship between Pb-B and P5N activity or purine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted)close to regressionlines show 95% confidence ranges of the regression line, and outmost curves (dotted) indicate 95% predictive intervals of individual values. (A) = P5N log y = 0.00726~ + 1.28, (B)=ATP log y = 0.00245~ 4.89, and (C)=AMP y =
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Fig. 2. Decay curves for nucleotidedegradation in blood from a lead worker during storage at 4OC after sampling. O = U D P C (log y= -0.000758~ 2), O=CDPC (log y = -0.00170~ 2), .=CTP (log y = 0.021lx 2),and O=ATP (log y = -0.0282~ 2).
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September/Odober 1990 [Vol. 45 (No. 5)]
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than 7 pmole/h-g Hb, which corresponds to a Pb-B concentration of 60 pg/lOO g (Fig. 3A). The elevated levels (mean standard deviation) of UDPG, CTP, and CDPC were 4 096 1 252,l 062 f 508, and 928 ? 319 nmol/dl, respectively, in 6 workers who Pb-6 levels exceeded 60 pg/lOO g. The mean concentrations ? standard deviations of UDPG, CTP, and CDPC in lead workers (n = 40)whose Pb-B were less than 20 pg/lOOg were 2 200 888,266 362, and 472 247 nmol/dl, respectively. Concentrations of ATP correlate negatively with PbB, whereas AMP concen-
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275
tration correlates positively (Fig. 38 and C). The correlation of ATP vs. Pb-B, both of which were corrected by Hct, was also siginificant (data not shown). In 47 lead workers, energy charge (y) was calculated from the equation, (ADPI2
+ ATP)/(AMP + ADP + ATP)
X
100 (Yo),
and was significantly decreased as Pb-B increased ( x ) (y = - 0 . 0 6 6 ~ 93.7, r = 0.557). These results suggest that lead affects pyrimidine nucleotide metabolism and purine nucleotide metabolism (energy production system). The ATP levels of 40 lead workers whose Pb-B were less than 20 bg/lOOg were 64 600 2
+
8 573 nmol/dl (nucleotides were extracted 4 h after sampling of blood). Discussion
Using anion-exchange HPLC to study lead-treated animals, Angle et a1.6 found three nucleotides (i.e., UTP, CTP, and CDP) that were increased in blood cells. In the present study, 14 species of nucleotides were separated, including CDPC and UDPG, which are the major nucleotides that are correlated with depression of P5N activity in lead-exposed subjects. This i s the first study that reports the composition of
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Fig. 4. Relation between Pb-B and pyrimidine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted) close to regression lines show 95% confidence ranges of the regression line, and outmost curves (doffed) indicate 95% predictive intervals of individual values. (A) = UDPC y = 3 3 . 7 ~ 782, (B) = CTP y = 1 3 . 6 ~- 307,and (C)=CDPC y = 8 . 3 8 ~ 119.
+
300
125
Fig. 5. Relationship between P5N activity and pyrimdine nucleotide levels. Solid lines or curves in center show the regression. Curves (dotted)close to regression lines show 95% confidence ranges of the regression line, and outmost curves (dotted) indicate 95% predictive intervals of individual values. (A) = UDPC log y = 0.0001 55x 1.32, (B) = CTP log y = - 0.000395~ 1.08, and (C) = CDPC 1- y = - 0.000492~ 1.21.
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Archives of Environmental Health
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Table 1.-Erythrocyte Nucleotide Concentrations in 47 Lead Workers and Their Correlations vs. Blood lead Concentration (Pb-B) or Pyrimidine 5-Nucleotidase (P5N) Activity
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Concentration (nmol/dl blood) Nucleotides
Mean
CDPC CMP UMP AMP GMP UDPC CDP UDP ADP GDP CTP UTP ATP CTP
472 19 51 1640 109 2 210 33 208 10 020 1012 266 956 62 600 2 981
SO 246 36 43 935 103
888 36 187 1 850 169 362 286 10 400 386
Correlation coefficients Pb-B VS. nucleotides 0.744 0.278 0.618 0.442 0.233 0.831
0.718 0.307 0.467 0.337 0.270
0.484
0.280 0.533 - 0.214 0.017 0.676 0.301 - 0.687 -0.393
0.617 - 0.191 0.016 0.823
0.406 -0.640 -0.369
abnormal nucleotides that accumulated in red blood cells of workers who were occupationally exposed to lead. The pyrimidine diphosphodiesters, CDPC and UDPG, and CTP are the most prominent abnormal nucleotides in blood cells of lead-exposed subjects. The nucleotides that were increased in lead workers are similar to those found in P5ND,7,8,9,but the concentrations are much lower than the massive amounts found in PSND. In the present study, we found that the levels of purine nucleotides (other than pyrimidine nucleotides) were also affected by lead exposure, suggesting disturbances of the energy production system by lead. As Pb-B values rose, ATP levels showed a slight reduction, whereas AMP levels were correlated positively with Pb-B (Fig. 3B and C). Energy charges were consequently decreased in workers who had Pb-B greater than 60 p.g/lOO g. Angle et a1,6,10 however, found that purine nucleotides were unchanged in red cells from lead-poisoned rabbits with mean Pb-B levels of 69.4 p.g/dl and in children with Pb-B levels between 30 and 72 p.g/dl. The energy charge was also normal in two children with P5ND.9 The observed pattern of glycolytic intermediates in lead poisoning also suggested that the activity of the EmbdenMeyerhof pathway was However, Torrance and Whittaker’ found that the concentrations of ADP and AMP in red blood cells of PSND were between 2 and 3 times normal, and the distribution of adenine nucleotides was abnormal with the lower energy forms being favored.
* * * * * * * * * * Submitted for publication August 7, 1989; revised; accepted for publication March 14,1990. Requests for reprints should be sent to: Tadashi Sakai, Ph.D., Center of Occupational Medicine, Tokyo Labor Accident Hospital, 13-21,Omoriminami-4, Ota-Ku, Tokyo 143, Japan.
!jeptember/October 1990 [Vol. 45 (No. 5)]
Pb-B VS. log nucleotides
0.800
P5N vs. nucleotides
log PSN vs. nucleotides
-0.591 0.051
-0.662 - 0.035 -0.556
-0.289 -0.064 - 0.603
- 0.345
- 0.448 - 0.444 - 0.395 0.227 0.232 - 0.638 -0.274
0.600 0.456
-0.051 -0.749 - 0.4% -0.439 0.256 0.208 -0.779 - 0.317 0.671 0.473
* * * * * * * * * * References 1. Sakai T, Araki T, Ushio K. Determination of pyrimidine 5’nucleotidase (P5N) activity in whole blood as an index of lead exposure. Br J Ind Med 1988;45:420-25. 2. Valentine WN, Fink K, Paglia DE, Harris SR, Adams WS. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5‘nucleotidase deficiency. J Clin Invest 1974;54:866-79. 3. Valentine WN, Tanaka KR, Paglia DE. Hemolytic anemia and erythrocyte enzymopathies. An Int Med 1985;103:245-57. 4. Tomoda A, Noble NA, Lachant NA, Tanaka KR. Hemolytic anemia in hereditary pyrimidine 5’-nucleotidase deficiency: nucleotide inhibition of G6PD and the pentose phosphate shunt. Blood 1982;60:1212-18. 5. Sakai T, Ushio K. A simplified method for determining erythrocyte pyrimidine 5’-nucleotidase (P5N) activity by HPLC and its value in monitoring lead exposure. Br J Ind Med 1986; 43 :839-44. 6. Angle CR, Stohs SJ, Mclntire MS, Swanson MS, Rovang KS. Lead-induced accumulation of erythrocyte pyrimidine nucleotides in rabbit. Toxicol Appl Phamacol 1980;54:161-67. 7. Torrance ID, Whittaker D. Distribution of erythrocyte nucleotides in pyrimidine 5’-nucleotidase deficiency. Br J Haematol 1979;43 ~423-34. 8. Swanson MS, Markin RS, Stohs SJ, Angle CR. Identification of cytidine diphosphodiesterase in erythrocytes from a patient with pyrimidine nucleotidase deficiency. Blood 1984;63:66570. 9. Ericson A, de Verdier CH, Hansen THR, Seip M. Erythrocyte nucleotide pattern in two children in a Norwegian family with pyrimidine S‘-nucleotidase deficiency. Clin Chim Acta 1983; 134%-33. 10. Angle CR, Mclntire MS, Swanson MS, Stohs SJ. Erythrocyte nucleotides in children-increased blood lead and cytidine triohomhate. Pediatr Res 1982:16:331-34. 11. M/wa lshida Y, Takegawa S; Urata G , Toyoda T. A case of lead intoxication: clinical and biochemical studies. Am J Hematoll981 ;1199-105. 12. Valentine WN, Paglia DE, Fink K. Lead poisoning. Association with hemolytic anemia, basophilic stippling, erythrocyte pyrimidine 5’-nucleotidase deficiency, and intraerythrocytic accumulation of pyrimidines. J Clin Invest 1976;58:926-32.
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