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The use of clozapine is limited by a relatively high incidence of drug-induced agranulocytosis. Clozapine is oxidized by bone marrow cells to a reacti...
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Chem. Res. Toxicol. 2008, 21, 869–873

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Testing the Hypothesis that Vitamin C Deficiency Is a Risk Factor for Clozapine-Induced Agranulocytosis Using Guinea Pigs and ODS Rats Julia Ip, John X. Wilson, and Jack P. Uetrecht* Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario M5S 3M2, Canada, and Department of Exercise and Nutrition Sciences, UniVersity at Buffalo, Buffalo, New York ReceiVed NoVember 9, 2007

The use of clozapine is limited by a relatively high incidence of drug-induced agranulocytosis. Clozapine is oxidized by bone marrow cells to a reactive nitrenium ion. Although many idiosyncratic drug reactions are immune-mediated, the fact that patients with a history of clozapine-induced agranulocytosis do not immediately develop agranulocytosis on rechallenge suggests that some other factor may be responsible for the idiosyncratic nature of this reaction. The reactive nitrenium ion is very rapidly reduced back to clozapine by vitamin C, and many schizophrenic patients are vitamin C deficient. We set out to test the hypothesis that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis using a vitamin C deficient guinea pig model. Although the vitamin C deficient guinea pigs did not develop agranulocytosis, the amount of clozapine covalent binding in these animals was less than we had previously observed in samples from rats and humans. Therefore, we studied ODS rats that also cannot synthesize vitamin C. Vitamin C deficient ODS rats also did not develop agranulocytosis, and furthermore, although covalent binding in the bone marrow was greater than that in the guinea pig, it was not increased in the vitamin C deficient ODS rats relative to ODS rats that had adequate vitamin C in their diet. Therefore, it is very unlikely that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis. Introduction Clozapine is an atypical dibenzodiazepine antipsychotic lacking extrapyramidal side effects. Despite its unique effectiveness in treating refractory schizophrenia, its use is limited because of the propensity to cause agranulocytosis in ∼0.8% of patients (1, 2). Agranulocytosis is a potentially fatal blood dyscrasia characterized by a dramatic decrease in neutrophil count. Therefore, patients on clozapine are required to monitor their neutrophil count weekly for the first 6–12 months after which the frequency of monitoring is often decreased (1). Little is known about the mechanism of drug-induced agranulocytosis and how clozapine causes this idiosyncratic drug reaction. Clozapine is bioactivated (oxidized) by activated human neutrophils and bone marrow cells to a reactive nitrenium ion through the myeloperoxidase-hydrogen peroxide system generating hypochlorous acid during the respiratory burst (3, 4). The idiosyncratic nature of this reaction suggests that it might be immune-mediated; however, the observation that it does not recur rapidly on rechallenge of patients with a past history of clozapine-induced agranulocytosis argues against, but does not completely exclude, this possibility (5). Studies have shown that the reactive metabolite of clozapine is capable of inducing apoptosis and cytotoxicity in neutrophils and bone marrow cells (6–10). It has also been shown that the nitrenium ion is capable of covalently binding to proteins in neutrophils and bone marrow tissue (11). Induction of apoptosis, cytotoxicity, and the formation of protein adducts by this reactive metabolite have been postulated to be associated with clozapine-induced agranulocytosis. Notably, the addition of ascorbate prevented detection * To whom correspondence should be addressed. Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Tel: 416-978-8939. E-mail: [email protected].

Figure 1. Clozapine is oxidized by the myeloperoxidase/H2O2/Clsystem of neutrophils forming the reactive nitrenium ion, which covalently binds to proteins. In the presence of vitamin C, the nitrenium ion is very rapidly reduced back to clozapine.

of clozapine oxidation by the myeloperoxidase-hydrogen peroxide system (12). We have demonstrated that the nitrenium ion is reduced back to clozapine by vitamin C (ascorbic acid) extremely rapidly such that we were unable to measure the rate (unpublished data). Neutrophils normally have high levels of vitamin C (in the millimolar range compared to the plasma level, which is only about 50 µM) (13). This is presumably because vitamin C is an antioxidant much needed to protect neutrophils from the oxidative stress generated by the large amounts of oxidants produced in these cells (14). There is even evidence linking the pathogenesis of schizophrenia with increased oxidative damage (15–17). Studies have shown that as many as 2% of institutionalized psychiatric patients might have vitamin C levels less than 0.1 mg/dL; detrimental effects on immunity are thought to occur at levels three times higher (18). Therefore, it is conceivable that vitamin C deficiency may be a major risk factor for clozapine-induced agranulocytosis because we know that vitamin C can detoxify the nitrenium ion (Figure 1). Indeed, ascorbic acid had been shown to attenuate clozapine-mediated

10.1021/tx7004025 CCC: $40.75  2008 American Chemical Society Published on Web 04/02/2008

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cytotoxicity toward HAS303 stromal cells and neutrophils presumably because of its ability to reduce the reactive metabolite and thus, resulting in a decrease in product adduct formation (6, 19). The present study evaluated the hypothesis that vitamin C deficiency is a risk factor for clozapine-induced agranulocytosis using guinea pigs and Osteogenic Disorder Shionogi (ODS1) rats as models. Both of these animals are unable to produce vitamin C because, similar to humans, they are both genetically deficient in l-gulonolactone oxidase required in the synthesis of ascorbic acid from glucose via the glucuronic pathway (20, 21). We also tested the hypothesis that depletion of intracellular vitamin C would result in an increase in clozapine-protein binding.

Materials and Methods Animals. Sixteen female Harley guinea pigs weighing approximately 700 g were purchased from Charles River (Montreal, QC), and 15 female ODS rats weighing approximately 200 g were purchased from CLEA Japan (Tokyo, Japan). The guinea pigs were housed in pairs in metal cages with paper bedding, and the rats were in pairs in plastic cages with corncob bedding in a 12:12 h light:dark cycle at 22 °C. They were acclimatized and given access to either standard guinea pig diet (guinea pigs) or rodent diet supplemented with vitamin C (ODS rats, Harlen Teklad, Madison, WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experiments performed on these laboratory animals were approved by University of Toronto’s animal care committee. Chemicals. Clozapine was provided by Novartis Pharmaceuticals Inc. (Dorval, QC). DL-Dithiothreitol (DTT), Ponceau S solution, 3,4-dihydroxybenzylamine (DHBA), and L-ascorbic acid were purchased from Sigma-Aldrich (Oakville, ON). Stock acrylamide solution (30%) and nitrocellulose were purchased from Bio-Rad (Mississuaga, ON). Horseradish peroxidase-conjugated goat antirabbit IgG (H+L chains) was purchased from Cedarlane (Burlington, ON). SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce (Rockford, IL). Vitamin C Deficient Diet and Clozapine Treatments. After acclimatization, all guinea pigs and rats were placed on the vitamin C-free purified rodent diet AIN-93G purchased from Harlen Teklad (Madison, WI). All animals were given drinking water that was deionized through a reverse osmosis system. Eight guinea pigs were placed in the vitamin C-adequate group (VN), and their diets were supplemented with ascorbic acid at 75 mg/kg/day given in the drinking water. The other eight guinea pigs were placed in the vitamin C-deficient group (VD) without ascorbic acid supplementation. In both the vitamin C-deficient and adequate groups, four animals (VNCL and VDCL) were dosed with clozapine at 50 mg/ kg/day in the drinking water starting on day 28 of the study and treated for 55 days. The rats were divided into 3 treatment groups: vitamin C-adequate (VN); vitamin C-deficient A (VD-A); vitamin C-deficient B (VD-B) each receiving a different level of vitamin C supplementation in their drinking water (1.67 g/L, 0.33 g/L, 0.2 g/L). In each of these groups, 3 of the 5 rats (VD-ACL and VDBCL) were dosed with clozapine at 50 mg/kg/day in their diet starting on day 28 of the study and treated for 42 days. This dose is approximately 5 times the therapeutic dose; higher doses are not well tolerated by the animals. Blood Collection and Leukocyte Counts. Blood samples were collected from each animal once a week. A sample of blood (200 µL) was obtained with a 25 G needle from the saphenous vein of the guinea pig or the tail vein of the rat and collected into Microvettes CB 300 Kalium-EDTA capillary tubes (Sarstedt, Montreal, QC). Total leukocyte counts were performed by mixing 10 µL of the blood sample with Turk Blood Diluting Fluid (Ricca 1 Abbreviations: ODS, Osteogenic Disorder Shionogi; VN, diet containing an adequate amount of vitamin C; VD, vitamin C-deficient diet; CL, clozapine cotreatment; DHBA, 3,4-dihydroxybenzylamine.

Ip et al. Chemical Co., Arlington, TX) at 1:9 and using a hemocytometer. Leukocyte differentials were obtained by preparing blood smears on slides and stained with Wright-Giemsa stain (Fisher Scientific Co., Middletown, VA). Peripheral neutrophil counts were calculated by multiplying the total leukocyte counts by the percentage of neutrophils in each blood sample. Collection of Bone Marrow and Liver. After 55 days and 42 days of clozapine treatment in the guinea pigs and ODS rats, respectively, all animals were sacrificed with an overdose of anesthetic (ketamine /xylazine). The femurs and tibia were removed, and bone marrows were collected by flushing with 20 mL of RPMI 1640 culture medium (University of Toronto, Tissue Culture). The bone marrows were resuspended by a five times passage through a 1 mL serological pipet tip. The cell suspension was centrifuged at 125g for 6 min. The red blood cells were then removed by resuspension of the cell pellet in red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA) for 6 min and centrifuged at 125g for 6 min. Tissue debris was removed by passing the cell suspension through a 70 µm nylon cell strainer (BD Biosciences, Bedford, MA) upon resuspension in phosphate-buffered saline (PBS; University of Toronto, Tissue Culture). The bone marrow cells were washed again in PBS and resuspended in 500 µL of cell lysis buffer (10 mM Tris-Cl at pH 7.4, 1 mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail). Livers were excised from the guinea pigs and stored at -80 °C. Liver tissue homogenate was prepared by taking a small aliquot of the frozen liver and homogenizing it in cell lysis buffer using a tissue homogenizer (9500 rpm, 3 bursts of 10 s). Bone marrow cell lysate and liver tissue homogenate samples were analyzed for protein concentration using BCA protein assay kit from Pierce (Rockford, IL). SDS-PAGE and Immunoblotting. Bone marrow cell lysate and liver tissue homogenate samples were diluted to a final protein concentration of 1 µg/µL with cell lysis buffer. One part of a 6× SDS-PAGE sample buffer (0.35 M Tris-Cl, 10% SDS, 4% glycerol, 0.02% bromophenol blue, and 18 mg/mL DTT) was added to 5 parts of sample. They were then heated at 90 °C for 10 min. SDS-PAGE was performed using a mini-gel system (MiniPROTEAN II, Bio-Rad). Stacking and resolving gels were 4% and 10% acrylamide, respectively. Prestained broad range molecular mass makers were used (Bio-Rad). A sample (20 µL) was loaded into each well. Gels were run at 120 V for 90 min until the dye front reached the bottom of the resolving gel. Electrophoretic transfer to nitrocellulose membrane was carried out at 100 V for 60 min using a mini Trans-Blot transfer cell (Bio-Rad) in transfer buffer (25 mM Tris-Cl, 0.19 M glycine, 20% methanol, 0.1% SDS). The nitrocellulose membrane was stained with Ponceau S solution for 5 min to assess the efficiency of the transfer. Lane densitometry was performed on the Ponceau S-stained blots. Only blots with all lanes having a net arbitrary lane density differing no more than 10% of the mean density of all lanes were used. The membrane was destained with wash buffer (100 mM Tris-Cl, 0.9% NaCl, 0.1% Tween 20). The subsequent steps were conducted at room temperature with gentle shaking on a rocker. The membrane was blocked with 5% (w/v) skimmed milk powder in 100 mM Tris-HCl buffer (pH 7.5) containing 0.9% NaCl and 0.1% Tween 20 for 1 h. The blocked membrane was then incubated for 15 h with an anticlozapine antibody diluted (1:3000 for liver tissue homogenate blots and 1:2000 for bone marrow cell lysate blots) in the Tris-HCl buffer; production of the anticlozapine antibody has been described previously (8). The membrane was washed with wash buffer for 10 min 5 times to remove any unbound antibodies. It was then incubated for 2 h with horseradish peroxidase-conjugated goat antirabbit IgG (H + L chain) antiserum diluted 1: 20 000 with wash buffer. After washing 5 times with wash buffer to remove any unbound antibodies, the membrane was incubated in SuperSignal West Pico Chemiluminescent Substrate for 10 min. The chemiluminescence on the blot was immediately captured using

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Table 1. Plasma, Serum, Peripheral Leukocytes, Bone Marrow Cells, and Heart Tissue Ascorbate Levels of Guinea Pigs in Each Treatment Group after 55 Days of Clozapine Treatmenta plasma (µM)

serum (µM)

leukocytes (nmol/106 cells)

bone marrow cells (nmol/106 cells)

heart (nmol/mg)

ND

ND

0.09 ( 0.03b

0.04 ( 0.01b

ND

ND

ND

0.06 ( 0.01

0.03 ( 0.01

0.72 ( 0.12c

150.8 ( 46.6

141.3 ( 44.6

0.69 ( 0.16

0.74 ( 0.10

74.1 ( 12.7

132.3 ( 81.5

130.7 ( 63.3

0.67 ( 0.26

0.60 ( 0.07

44.0 ( 21.2b

Vitamin C-Deficient Diet VD with clozapine treatment VDCL Vitamin C-Adequate Diet VN with clozapine treatment VNCL

c

c

a

Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection. Assay sensitivity was 2 pmol. ND denotes nondetectable levels. Values are expressed in mean ( SD in each treatment group (n ) 4). b p < 0.05 compared to treatment group VN. c p < 0.05 compared to treatment group VNCL.

the FluorChem8800 imaging system by Alpha Innotech (San Leandro, CA) by exposing it for 10 min to visualize bound antibodies. Vitamin C Status Assessment. Upon sacrifice of each guinea pig, blood was obtained by cardiac puncture for serum, plasma, and peripheral leukocytes isolation; liver, heart, and skeletal muscle were excised; and bone marrow cells were isolated for ascorbate level assessment in each of these organs. Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection according to a previously described procedure (22). Organ samples were homogenized (Kontes Pestle Motor Mixer) at 4 °C in a metaphosphoric acid solution (8.5 g/L) that contained 3,4-dihydroxybenzylamine (DHBA) as an internal standard. Plasma and serum samples were extracted with equal volumes of methanol with 200 uM DHBA. The homogenates or extracts were then centrifuged at 4 °C, passed through a 45 um Millex filter and then injected into the HPLC system. A Resolve C18 90 A silica 3.9 × 150 mm column was used with a flow rate of 0.5 mL/min. The mobile phase contained 80 mM sodium acetate, 0.015% metaphosphoric acid, 1 mM n-octylamine, and 15% methanol (pH 4.6). Ascorbate and DHBA in samples and standards were quantified with a Waters M460 amperometric detector. Assay sensitivity was 2 pmol. The ascorbate concentrations of samples were determined by interpolation on an external standard curve and corrected for DHBA recovery. Blood and organ tissues were taken from two guinea pigs, one on the vitamin C-deficient diet and one from the vitamin C-adequate diet, after 28 days on the diet and from the rest of the guinea pigs at the end of the study. Blood was taken from each rat at day 28 of the study for serum ascorbate level assay prior to clozapine treatment. Statistical Analysis. Statistical analyses were performed using the GraphPad Prism 4 software (GraphPad Software Inc.). Unpaired t-tests (two tailed, 95% confidence interval) were used to compare between treatment groups; values of p e 0.05 were considered statistically significant.

Results Vitamin C Status. In a preliminary study, the effect of 28 days of the vitamin C-deficient diet was determined. Ascorbate concentrations in the guinea pig given the deficient diet were an order of magnitude less than a control given a normal vitamin C diet, and the plasma, serum, heart, and skeletal muscle ascorbate concentrations were actually below the detection limit (data not shown). Ascorbate levels remained very low in the guinea pigs on the vitamin C-deficient diet both with and without 55 days of clozapine treatment compared to those on the vitamin C-adequate diet (p < 0.05; Table 1). Although there was a trend to lower ascorbate levels in clozapine-treated animals in all tissues and with both vitamin C deficient and adequate vitamin C diets, the difference was only significant in the heart. In the ODS rats, after 28 days the serum ascorbate levels of animals

Table 2. Serum Ascorbate Levels of ODS Rats after 28 Days of Vitamin C Supplemented Diet Prior to Clozapine Treatmenta serum ascorbate concentration (µM) VD-A VD-B VN

10.82 ( 8.42b 5.83 ( 2.96b 29.24 ( 15.62

a Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection. Assay sensitivity was 2 pmol. Values are expressed in mean ( SD in each treatment group (n ) 5). The VN group received vitamin C through drinking water at 1.67 g/L, VD-A at 0.33 g/L, and VD-B at 0.2 g/L. b p < 0.05 compared to treatment group VN.

Figure 2. Peripheral neutrophil counts of guinea pigs during clozapine treatment. Values are expressed as the mean ( SD of each treatment group, n ) 4.

on the lowest vitamin C diet were only one-fifth of that of animals receiving the vitamin C-adequate diet (Table 2). Peripheral Leukocyte Counts. Total leukocytes and peripheral neutrophil counts were within the normal ranges of guinea pigs and rats in these studies (23, 24). Significant changes in total leukocyte and peripheral neutrophil counts were not observed during the course of clozapine treatment in either the group of guinea pigs or rats given an adequate or deficient vitamin C diet (Figures 2 and 3). Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins. Covalent binding of clozapine to hepatic proteins in the liver homogenate and bone marrow proteins in the bone marrow cell lysate were detected in all guinea pigs and rats dosed with the drug. However, the binding in samples from the guinea pigs produced only faint bands ranging from 7.2 to 203 kDa (Figures 4 and 5). In contrast, immunoblots of the rat liver homogenate showed bands with molecular masses ranging from 30 to 240 kDa (Figure 6), whereas immunoblot

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Figure 3. Peripheral neutrophil counts of ODS rats during clozapine treatment. Values are expressed as the mean ( SD of each treatment group, n ) 4.

Figure 6. Immunochemical detection of clozapine-modified hepatic proteins from clozapine-treated vitamin C-deficient ODS rats given drinking water supplemented with L-ascorbic acid at 1.67 g/L (VNCL), 0.33 g/L (VD-ACL), and 0.2 g/L (VD-BCL). Rats were administered a daily dose of 50 mg/kg clozapine for 42 days. Protein loading was 30 µg/lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents a sample from an individual animal. An untreated control sample (VN) was analyzed on a separate blot and did not show any clozapine protein covalent binding.

Figure 4. Immunochemical detection of covalent binding of clozapine to hepatic proteins of vitamin C-deficient guinea pigs (VDCL) and those that received adequate vitamin C (VNCL), given a daily dose of 50 mg/kg clozapine for 55 days. Lane VN represents a sample from a guinea pig on the vitamin C-adequate diet without clozapine treatment. Protein loading was 10 µg/lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents a sample from an individual animal.

Figure 7. Immunochemical detection of clozapine-modified bone marrow proteins from clozapine-treated vitamin C-deficient ODS rats given drinking water supplemented with L-ascorbic acid at 1.67 g/L (VNCL), 0.33 g/L (VD-ACL), and 0.2 g/L (VD-BCL). Rats were administered a daily dose of 50 mg/kg clozapine for 42 days. Protein loading was 30 µg/lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents a sample from an individual animal. An untreated control sample (VN) was analyzed on a separate blot and did not show any clozapine-protein covalent binding.

Discussion

Figure 5. Immunochemical detection of covalent binding of clozapine to bone marrow proteins of vitamin C-deficient guinea pigs (VDCL) and those that received adequate vitamin C (VNCL), given a daily dose of 50 mg/kg clozapine for 55 days. Lane VN represents a sample from a guinea pig on the vitamin C-adequate diet without clozapine treatment. Protein loading was 60 µg/lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents a sample from an individual animal.

of the bone marrow cell lysate blot showed only one prominent band at 49 kDa (Figure 7). From previous studies, we believe that this band represents myeloperoxidase, the enzyme responsible for reactive metabolite formation. There was no significant difference in the amount of clozapine covalent binding in samples from guinea pigs or rats between animals given an adequate vitamin C diet and those on the deficient diet.

In an in Vitro study conducted by Pereira et al. the addition of ascorbic acid significantly decreased bone marrow stromal cell death caused by clozapine (6). This is likely due to the ability of ascorbic acid to inhibit clozapine oxidation or reduce the reactive nitrenium ion back to clozapine (12). Although scurvy is not common in present day society, schizophrenic patients have been reported to have low to scorbutic levels of vitamin C for reasons such as poor diets that resulted in nutritional deficiency (18, 25). It has been suggested that coadministration of vitamin C with clozapine may reduce risk of agranulocytosis by its cytoprotective properties (26). The aim of this study is to evaluate the hypothesis that vitamin C deficiency is a risk factor for clozapine-induced agranulocytosis in guinea pigs. Besides humans and other primates, the guinea pig is one of the few species that cannot produce vitamin C. As expected, ascorbate levels in plasma, serum, peripheral leukocytes, bone marrow cells, and heart were much lower or even below the detection limit in guinea pigs given a vitamin C-deficient diet (Table 1). Despite prolonged treatment of vitamin C-deficient guinea pigs with clozapine, decreasing trends in white blood cell or neutrophil counts were not observed (Figure 2). However, the degree of covalent binding observed in guinea pig samples (Figures 4 and 5) was much less than we had observed previously in samples from rats treated with

Vitamin C and Clozapine-Induced Agranulocytosis

clozapine or neutrophils from humans treated with clozapine, suggesting the degree of bioactivation in guinea pigs is less than that in rats and humans. Therefore, we judged that the guinea pig is not a good animal in which to test the hypothesis. Normal rats cannot be made vitamin C-deficient because they are capable of producing ascorbic acid from glucose via the glucuronic pathway. However, a mutant strain, the ODS rat, bears an inborn deficiency of 1-gulonolactone oxidase and thus it lacks the ability to produce ascorbic acid. These animals can be given a marginally scorbutic diet without showing any overt signs of scurvy for a few months. Therefore, this animal offered a good alternate to the guinea pig for these studies because we had already studied covalent binding of clozapine in rats, and covalent binding of clozapine in rat neutrophils is comparable to that in human neutrophils. As with guinea pigs, the serum levels of ascorbate in ODS rats on a vitamin C-deficient diet were significantly lower compared to those measured in vitamin C-adequate group or in Wistar rats (Table 2). However, as with guinea pigs, there was no effect of the vitamin C deficient diet on neutrophil counts (Figure 3). Furthermore, vitamin C deficiency did not appear to have any significant effect on the amount of clozapine covalent binding in either the hepatic or bone marrow tissues from guinea pigs or ODS rats (Figures 4-7). It is interesting that clozapine treatment appeared to decrease ascorbate levels in the heart and it is possible that this is related to the myocarditis sometimes observed in patients treated with clozapine (27, 28). Given the very rapid inactivation of the nitrenium ion by vitamin C, it is somewhat surprising that vitamin C deficiency did not lead to an increase in covalent binding. This suggests that other detoxication pathways for the nitrenium ion exist. It has been shown that a decrease in glutathione in rats can induce a compensatory increase in ascorbic acid level (29). If the reverse is also true it may explain why covalent binding was not affected by vitamin C deficiency. If vitamin C deficiency had failed to lead to agranulocytosis in this model but did significantly increase covalent binding, it would suggest that vitamin C deficiency might be necessary but not sufficient to lead to clozapine-induced agranulocytosis. However, the observation that vitamin C deficiency did not even significantly increase covalent binding of clozapine makes it very unlikely that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis. Acknowledgment. J.P.U. is the recipient of the Canada Research Chair in Adverse Drug Reactions. This work was supported by grants from the Canadian Institutes of Health Research.

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