C. C. WILLHITE'I "a, W. C. HAWKES~, S. T. OMAYE§, W. N. CHOY[[, D. N. Cox¶ and ... Western Human Nutrition Research Center, Presidio of San Francisco,.
i
Fd Chem. Toxic. Vol. 30, No. I 1, pp. 903-913, 1992 Printed in Great Britain. All rights reserved
0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
Research Section ABSORPTION, DISTRIBUTION A N D ELIMINATION OF SELENIUM AS L-SELENOMETHIONINE IN N O N - H U M A N PRIMATES* C. C. WILLHITE'I"a, W. C. HAWKES~, S. T. OMAYE§, W. N. CHOY[[, D. N. C o x ¶ and M. J. CUKIERSKI**b tDepartment of Toxic Substances Control, California EPA, 700 Heinz Street, Suite 200, Berkeley, CA94710, ~USDA-ARS Western Human Nutrition Research Center, Presidio of San Francisco, CA94129, §Department of Nutrition, Fleischmann Bldg, Rm 113, University of Nevada, Reno, NV 89557, IlSchering-Plough Corporation, PO Box 32, Lafayette, NJ 07848, ¶ENVIRON Corporation, 5820 Shellmound Street, Suite 700, Emeryville, CA 94608 and **California Primate Research Center, University of California Davis, CA 95616, USA (Accepted 29 June 1992) Abstract--20 adult female macaques (Macaca fascicularis) were given oral doses of L-selenomethionine (L-SeMet) equivalent to 0, 25, 150, 300 and 600 #g selenium (Se)/kg body weight, and plasma, erythrocyte, hair, faecal and urine Se concentrations were determined. The macaques were scheduled for 30 daily oral doses of L-SeMet, but systemic toxicity necessitated dose reduction in several animals; two macaques given 600#g Se/kg body weight/day for 10-15 days died, and the concentration of Se in their tissues was determined and compared with Se concentrations in tissues collected from one untreated animal. Circulating and urinary Se concentrations in control macaques were within the normal human ranges. Plasma, erythrocyte, hair and urinary Se concentrations were generally dependent on the dose of L-SeMet administered. Plasma Se reflected more immediately exposure to L-SeMet, whereas erythrocyte Se concentrations increased and decreased more slowly. In some cases, erythrocyte Se was still increasing or showed a plateau after L-SeMet treatment was discontinued. Plasma Se concentrations of 6.7-7.3 ppm were observed in the two animals that died due to acute toxicity to L-SeMet. Neither plasma nor erythrocyte GPx activity was influenced by a single L-SeMet dose, but an increase in erythrocyte GPx activity occurred with continuous exposure. Total tissue Se increased 13-28-fold in macaques given 600/zg Se/kg body weight/day for 10-15 days, with the liver and kidneys containing the highest Se concentrations.
INTRODUCTION
Selenium (Se) and various Se-containing compounds have been regarded as cancer chemopreventive agents in animals and humans for at least 70 years (Combs and Combs, 1986). Prophylactic Se supplementation has been recommended for clinical cancer chemoprevention (Chen and Clark, 1986; Dimitrov and Ullrey, 1986; Reinhold et al., 1989; Shamberger, 1970; Yu et al., 1990) based in part on the numerous supporting animal studies (Lo and Sandi, 1980; Shamberger, 1970; Shapiro, 1972). However, the potential utility of Se in the prevention of human cancer is limited by its toxicity. Despite *Presented in part at the 30th Annual Meeting of the Society of Toxicology, Dallas, TX (Toxicologist 1991, lt, 93). "To whom correspondence should be addressed. bpresent address: ALZA Corporation, 950 Page Mill Road, Palo Alto, CA 94304, USA. Abbreviations: AUC =area under the curve of concentration against time; Cm,. = peak concentration; GPx = glutathione peroxidase; L-SeMet = L-selenomethionine; Se=selenium; h/2a=absorption biological half-life; tl.,2e = elimination biological half-life; Tm~~ = peakconcentration time; TWA = time weighted average.
ample evidence for Se essentiality in human nutrition (Stadtman, 1990), excessive consumption of Se can cause mucocutaneous disorders, oedema, nausea, anorexia, emesis, diarrhoea, fatigue, incoordination, paralysis, hemiplegia and death (Lo and Sandi, 1980; Olson, 1986; Yang et al., 1983). A m o n g the numerous forms of Se, L-selenomethionine (L-SeMet) has been suggested to be a relatively safe c o m p o u n d for use in clinical trials (Willett and Stampfer, 1986). Preclinical studies of L-SeMet have been initiated by the US National Cancer Institute to evaluate its toxicity and pharmacokinetics in rats and dogs (NCI, 1990). Since L-SeMet is currently under investigation as a potential anti-cancer drug, there is a need to determine toxic exposure levels and the dispositional parameters that govern the regulation of pharmacological doses in the body. The present study deals with the uptake, distribution and elimination of Se in female long-tailed macaques (Macaca fascicularis) treated orally with L-SeMet for 30 consecutive days. The data presented here were based on a range-finding toxicity study (Cukierski et al., 1989) conducted to design a teratology bioassay of L-SeMet in primates (Tarantal et al., 1991). Plasma, erythrocyte, hair, faecal and
903
C . C . WILLHITE et al.
904
urinary Se together with plasma and erythrocyte glutathione peroxidase (GPx; EC 1.11.1.9) specific activities were measured at regular intervals during and after dosing. Total Se in selected tissues of those macaques that died as a result of Se intoxication were compared with tissues from control macaques. The results are discussed in relation to the kinetics of L-SeMet in women and in the light of the doses selected for clinical trials. MATERIALS AND M E T H O D S
Chemicals. L-SeMet (2-amino-4-[methylseleno]butyric acid; 99% pure) was purchased from Sigma Chemical Co. (St Louis, MO, USA). The compound was stored in the dark at - 2 0 ° C . All solvents and other analytical agents were reagent grade. Animals. Macaques were housed and maintained in accordance with the standards established by the Animal Welfare Act (1966) and the Institute for Laboratory Animal Resources (Bethesda, MD, USA). The protocol was reviewed and approved by the Institutional Animal Care and Use Committee, and all procedures were consistent with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals (1986). Detailed descriptions of housing, diet, handling and special treatment have been reported by Cukierski et al. (1989). Se concentrations in the stock diet
(159 4- 38 ppb) and drinking water (0.61 _ 0.09 ppb) were measured using the method of Watkinson (1966) and accounted for an average daily dietary Se intake lower than or equivalent to l l # g Se/animal/day. Selected animals (nos 1, 3, 5, 6, 8 and. 14; Table 1) received Sustagen (Mead Johnson & Co., Evansville, IN, USA) or fruit to prevent anorexia. The Se contribution from this treatment was not measured but was not considered substantial in comparison with the quantities of L-SeMet administered since these supplements were given for an abbreviated period or only limited amounts were supplied. Experimental protocol. 20 adult female long-tailed macaques (Macaca fascicularis; 2.2-5.7kg) were randomly assigned to one of five treatment groups (0, 25, 150, 300 or 600#g Se/kg body weight/day). The animals were initially scheduled for 30 daily oral doses at the assigned levels, but because of toxicity, doses were reduced for some macaques given 300 or 600/lg Se/kg body weight/day (Table I). Following dosing on day 15, animal no. I was removed from the study and euthanasia was performed for ethical reasons two days later; animal No. 2 died before dosing on day 11 as a result of acute Se toxicity. Time-weighted-average (TWA) doses for the 17 animals that completed 30 days of L-SeMet treatment are shown in Table 1. Solutions of L-SeMet were freshly prepared daily in distilled, deionized water by
Table I. Doses of L-selenomethionine (L-SeMet) and duration of exposure used in the multiple-dose study TWA dose (#g L-SeMet/kg body weight/day)
Animal no.
600 600 300 300 300 300 300 203
1" (20824) 2~(20828) 3 (22027) 4 (22747) 55§(23251) 6~(23410) 7 (22359) 8§(18156)
188
9 (20289)
190
10 (20830)
150 150 150 117
11§(19345) 12 (22768) 13 (17862) 14 (20919)
62
15§(18213)
25 25 0 0 0
16 (20916) 17§(20825) 18§(20918) 19 (22113) 20~(20921)
Dose of L-SeMet administered (/lg/kg body weight/day)
Treatment days
600 600 300 300 300 300 300 600 400 150 600 400 150 300 25 150 150 150 300 25 300 25 25 25 0 0 0
I 15 1-10 I 30 1 30 1-19 1-30 I 30 I 3 4 5 30 1 2 3 4-30 1-18 19 30 1-30 1-30 1-30 1-10 11 30 14 5-30 1-30 I 30 1-30 1 30 1-30
TWA dose = time-weighted-average dose *Macaque removed from the study on day 15 due to severe intoxication and killed on day 17 for ethical reasons. "t'Macaque no. 2 died suddenly on day I I. SMacaque removed from the study on day 19 due to severe intoxication. §Macaques included in day-I blood sampling following a single oral intubation of L-SeMet at the indicated dose. Numbers in brackets are animal code numbers from the California Primate Research Center.
Selenium pharmacokinetics high-shear mixing and were administered immediately after preparation. For the single-dose phase of the study, macaques nos 5, 6, 8, 11, 15, 17, 18 and 20 were acclimatized to primate restraint chairs prior to initiation of the study. No untoward behaviour was observed during sample collection. Subsequent to chair restraint, a urinary catheter was inserted followed by administration of 0-4500/~g Se/kg body weight as L-SeMet by way of nasogastric intubation at a volume of 5 ml/kg body weight. Macaques were kept in restraint chairs from 0 to 6 hr post-intubation and then placed in stainless-steel metabolism cages. Blood was collected by venipuncture at 0, 0.25, 0.5, 1, 3, 6, 12 and 24 hr after L-SeMet intubation. Urine was collected by catheter between 0-3 and 3-6 hr and from metabolism cage pans between 6-12 and 12-24hr. Faeces were collected between 0-12 and 12-24 hr after treatment. In the multiple-dose phase of the study, macaques received 0-600/~g Se/kg body weight as L-SeMet each day for up to 30 consecutive days. Animals were restrained for blood collection immediately before intubation with L-SeMet on days 1, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 43 (2wk after the final dose). In selected macaques, blood was also collected at 0.25, 0.5, I, 3, 6, 12 and 24 hr after dosing on days 15 and 30; 1 ml was collected through a syringe and transferred immediately into an EDTA Vacutainer (Becton Dickinson, Rutherford, N J, USA). More frequent blood sampling was not feasible because of limitations on the total quantity of blood that could be collected. Blood was separated by centrifugation and the plasma decanted; erythrocytes were resuspended in isotonic saline and then collected as a pellet after centrifugation. Hair samples were clipped from a 5-cm 2 area of the dorsal subscapular region from each animal just before the first dose of L-SeMet; hair was collected again from the same areas on days 22 (or 23), 39 and 43 (or 44), and was then stored in plastic bags at room temperature until analysis. Tissues from the two macaques that died were collected at autopsy and frozen. Plasma, erythrocytes, urine, faeces and tissues were stored at - 7 0 ° C in the dark until analysis. Selenium analyses. Quantitative determination of total Se was carried out using a modification of the spectrofluorometric method of Watkinson (1966). Plasma, erythrocytes (0.25 ml), urine (0.1-2 ml), tissue (0.1--0.5 g), faeces (0.1-0.5 g) or hair samples were mixed with 1 ml concentrated perchloric acid and 2.5 ml nitric acid. The samples were incubated for 90 min at 140°C and digested at 200°C for 75 min. Selenates in various samples were reduced to selenites after addition of 1 ml HCI (4N) and incubated at 150°C for 15 min. 1 ml 2 M-glycine-0.09 M-EDTA and 4 drops of 0.02% cresol red were added, and the pH was adjusted to 1.5-2.0 with 7M-NH4OH. 1.5ml glycine (2 M, pH 1.75) was added and the samples were diluted to 8 ml with distilled H20. 1 mi recrystallized 2,3-diaminonaphthalene [Sigma; 0.25% (w/v) in 0.1 N-HCI] was added, and the solution was
905
incubated at 50°C for 45 min. The solution was then extracted in 3 ml cyclohexane and the fluorescence of the resulting piazselenoi was measured with a fluorescence spectrometer (LS-3B; Perkin-Elmer, Norwalk, CT, USA) at 370 nm excitation and 518 nm emission. Analytical standards for Se were obtained from the National Institute of Standards and Technology (formerly the National Bureau of Standards, NBS, Gaithersburg, MD, USA) as standard reference materials (NIST-SRM) 3149. Standard reference materials of bovine liver (NBS-SRM 1577 or NISTSRM 1577a), mixed diet (NIST-SRM 8431), urine (NIST-SRM 2670), drinking water (NIST-SRM 1643b) or quality-control samples of duplicate pooled human erythrocytes, plasma or urine were included in each measurement series. Glutathione peroxidase. The activity of glutathione peroxidase (GPx) in macaque plasma, erythrocytes and in tissues collected at autopsy was determined by the method of Hawkes and Craig (1990). Interassay variation was taken into account by including duplicate pooled plasma or erythrocytes in each assay and by comparison with the standard activity of bovine erythrocyte GPx (G6137, Sigma). Plasma and erythrocyte proteins were quantified (Hawkes and Craig, 1991) using bovine serum albumin as the standard. Enzyme activity was calculated as /~mol glutathione oxidized/min/g protein. Pharmacokinetics. The total endogenous plasma or erythrocyte Se concentration determined for each macaque prior to treatment was subtracted from all subsequent measurements. A simple one-compartment open model with first-order uptake and elimination (Ct = [DKa/{V(ka - ke)}] x [ - e x p ( - k e t ) exp(-k~t)]) was then used to plot the curves of Se concentration against time. The apparent absorption and elimination biological half-lives (h/2a and h/2e, respectively), the areas under the curve of concentration against time (AUC), peak concentration (Cm~x) and peak-concentration time (Tmax)were calculated (Gibaldi and Perrier, 1982; Nelder and Mead, 1973; Statistical Consultants Inc.; 1986). The rate of total urinary Se elimination was determined from the entire volume excreted at 0-3, 3-6, 6-12 and 12-24 hr. RESULTS
Single-dose analyses Endogenous plasma and erythrocyte Se concentrations for the three control macaques averaged 160 ___25 and 140 _ 25 ppb, respectively; the corresponding control values over the 30-day treatment period ranged from 85 to 237 and 109 to 196 ppb. Intubation of a single oral dose of L-SeMet at 25 #g Se/kg body weight in macaque no. 17 (Table 2) resulted in a prompt increase in plasma Se from 140 to 260 ppb with a Cm~xat 3.4 hr; thereafter, plasma Se declined only slightly to 225 ppb at 24 hr. Over the same period, erythrocyte Se increased from 99 to
906
C. C. WILLHITE et al. Table 2. Kinetic parameters of selenium following administration of a single oral dose of L-selenomethionine (L-SeMet) in macaques
Dose (/~g Se/kg body weight)
Animal no.
AUC ( P g ' hr/ml)t
25 150 300 300 300 600
17 I1 15 5 6 8
2 9 23 20 15 49
25 150 300 300 300 600
17 I1 15 5 6 8
0.68 1.5 2.2 0.83 0.88 2.7
h/2a (hr)~
Plasma parameter* 0.5 1.4 2.3 0.5 6.2 0.9 Erythrocyte parameler* 1.5 0.33 1.8 0.1 0.45 0.42
tvze (hr):~
T,,,.,, (hr):~
Cm,x(/ag/ml):~
42 I 18 21 21 6 33
3.4 8.9 8.1 2.7 8.8 4.9
0.1 0.4 1.2 1.1 0.8 2.5
2.6 16 50 97 23 15
2.8 1.9 9.0 0.8 2.6 2.3
0.03 0.05 0.11 0.04 0.05 O. 17
*Parameters are as defined in Materials and Methods. Values are for individual macaques numbered as in Table 2. tCalculated by integration of the curve of Se concentration against time from 0 to 24 hr, using the trapezoidal approximation (Gibaldi and Perrier, 1982). +Parameters estimated by fitting non-linear least squares of the data on Se concentration against time to a one-compartment, single-dose model (plasma: R 2 = 0.88~).99; erythrocytes: R 2 = 0.714).99) (Nelder and Mead, 1973; Statistical Consultants, 1986).
170ppb at 24hr with a Cmax at 2.8 hr (Table 2); the concentration of Se in erythrocytes was equivalent to 11.4 + 9.6% of that in plasma at Cm,x. Following a dose of 150/~g Se/kg body weight, plasma Se increased from 152 to 580 ppb at 12 hr. After a single dose of 300 ~g Se/kg, mean plasma Se increased from 200 to 1177 ppb at 6 hr; after a single dose of 600ttg Se/kg, mean plasma Se increased from 200 to 2580 ppb at 3 hr. Despite a marked interindividual variation, plasma A U C and Cm,x generally increased with dose. In almost all animals, the rate of Se appearance in plasma was much greater than that of Se elimination; however, in macaque no. 6 these rates were equivalent. While certain erythrocyte parameters (e.g. Cm,x and AUC) tended to increase with dose, others remained more constant (Table 2). Elimination of Se from plasma and erythrocytes was much slower than its rate of appearance. Pharmacokinetic analyses were limited by the dosing and sampling protocols since all of the macaques were given a second dose of L-SeMet immediately after the 24-hr blood collection, when Se concentrations had only just begun to decline. Neither plasma nor erythrocyte GPx activity varied with time or dose following a single oral dose of L-SeMet (data not shown). The rate of urinary Se elimination increased with increasing L-SeMet doses (Fig. 1, Day 1). Control urine contained 6.4-74ppb Se. 25/~g Se/kg body weight increased urinary Se from 15 to 1360 ppb at 3-6 hr; at 150 or 300/~g Se/kg, urinary Se varied from 20-30 to approximately 3000 ppb in some animals. Urinary Se concentration was increased to more than 5400 ppb at 3-6 hr by 600/~g Se/kg body weight; even at 12-24hr after dosing, the rate of urinary Se elimination had not returned to pretreatment conditions (Fig. 1, Day 1). The mean rate of urinary Se elimination in controls was 0.12/~g/hr; in test animals, this rate was 1.22, 5.55, 3.23 and 6.12/ag/hr
over the first 24 hr following a single oral dose of 25, 150, 300 or 600#g Se/kg body weight, respectively. Faecal Se in control macaques (nos 18 and 20) averaged 240 __+94ppb. There was no increase in faecal Se at 0-12 or 12-24hr after intubation of 25 (no. 17) or 150#g Se/kg (no. 11) compared with controls. After treatment with 300~g Se/kg, faecal Se increased from 278 + 84ppb at 0-12 hr to 788 + 304 ppb at 12-24 hr. At 12-24 hr after administration of 600/~g Se/kg, the faecal Se concentration for macaque no. 8 was twice as high as the control value (515 ppb).
Multiple-dose analyses Protracted oral exposure to L-SeMet led to Se accumulation in macaque erythrocytes (Fig. 2A) and plasma (Fig. 2B). On discontinuation of oral L-SeMet intubation on day 30, plasma Se declined but erythrocyte Se either showed a plateau or decreased only very slowly. Plasma AUC and Cm,x values tended to increase with dose. The prolonged elimination of Se from plasma observed after a single dose of L-SeMet (Table 2) also occurred after treatment with multiple doses (Table 3). Plasma t~e was increased 6-32 times by multiple Se doses (Table 3) compared with single Se doses (Table 2). At 150/~g Se/kg, there was a 2.5-fold difference in rates of elimination of circulating Se between macaques given equivalent doses (Table 3). Plasma Tmax occurred at termination of treatment (day 30) (Fig. 2B). Kinetic parameters could not be estimated accurately in erythrocytes because Se concentrations showed continued accumulation (Fig. 3A,B,E), a plateau (Figs 2A and 3C,D,G) or only slight reductions (Figs 2A and 3F) at the last blood collection. Compared with plasma, the elimination of Se from erythrocytes was very slow and resulted in higher Se concentrations at termination of the study. In those macaques treated with reduced Se doses because of their poor physical condition, plasma
Selenium pharmacokinetics 12 -/
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I o I ~____ 6 12 18 Mean time after dosing (hr) Fig. I. (day 1) Rate of urinary Se elimination in female macaques given a single oral dose of distilled water (O) 25 (0), 150 (A), 300 (A) or 600 (I-q) #g Se/kg body weight as L-SeMet. Urine was collectedjust prior to dosing and at 0-3, 3-6, 6-12 and 12-24hr after intubation. Values represent the rates calculated from the entire volume of urine excreted by one animal treated with 0, 25, 150 or 600/ag Se/kg body weight and the mean ( + SE) from three animals treated with 300/~g Se/kg body weight. (day 15) Rate of urinary Se elimination in female macaques given a single oral dose of distilled water (O), 25 (O), 150 (A) or 300 (A) #g Se/kg body weight as L-SeMet on the morning of day 15 after treatment with identical doses for 14 consecutive days. Urine was collected just prior to dosing and at 0-3, 3-6, 6-12 hr and 12-24 hr after intubation. Values represent the rates calculated from the entire volume of urine excreted by one animal treated with 0, 25 or 150#g Se/kg body weight/day and the mean (+ SE) from two animals treated with 300#g Se/kg body weight/day. (day 30) Rate of urinary Se elimination in female macaques given a single oral dose of distilled water (O), 25 (0), 150 (A) or 300 (A) /ag Se/kg body weight as L-SeMet on the morning of day 30 after treatment with identical doses for 29 consecutive days. Urine was collected just prior to dosing and at 0-3~ 3-6, 6-12 and 12-24hr after intubation. Values represent the rates calculated from the entire volume of urine excreted by one macaque treated with each dose of Se.
Se reflected the more immediate exposure to Se and the changes in erythrocyte Se concentrations were delayed (Fig. 3). After 10 to 15 days of treatment with 600#g Se/kg body weight/day, total plasma Se increased from 0.2 to 6.7-7.3 ppm and erythrocyte Se increased from 0.19 to 5.67ppm (Fig. 3A,B). Plasma Se concentrations greater than 6ppm were observed in these two animals at death. For macaque no. 8 treated with 600#g Se/kg body
907
weight/day for the first 3 days (Table 1), plasma Se increased from 0.18ppm at the beginning of exposure to 5.2 ppm on day 6 (Fig. 3C); gastrointestinal disorders and loss of appetite were associated with the initial high doses, but when the dose was reduced to 150 #g Se/kg body weight/day plasma Se declined and these signs abated. Plasma Se declined after cessation of treatment, but erythrocyte Se continued to increase even after the dose was reduced. During 18 days of treatment with 300#g Se/kg body weight, plasma Se increased steadily in macaque no. 10 (Fig. 3D). Although the TWA dose for animal no. 8 (203 #g Se/kg body weight/day) was only 7% greater than that administered to macaque no. 10 ( T W A = 190#g Se/kg body weight/day), plasma AUC was 3 2 " greater in the former than in the latter (Table 3). Unlike macaque no. 8, where erythrocyte Se accumulation lagged behind plasma Se accumulation (Fig. 3C), erythrocyte and plasma Se in macaque no. 10 were parallel during the first week of L-SeMet intubation (Fig. 3D). After a 12-fold reduction in dose on day 19, erythrocyte Se began to decline in line with the reduction in plasma Se (Fig. 3D). A skin rash developed in macaque no. 9 when plasma Se concentrations reached approximately 4ppm (Fig. 3E). Plasma Se continued to increase even after dose reductions and began to decline only after cessation of treatment. In comparison with macaque no. 10 (Fig. 3D), high initial Se doses (600 pg Se/kg body weight/day) resulted in a 1.7-fold increase in AUC in macaque no. 9 despite the fact that the TWA doses for the two animals were very similar. In contrast to macaques nos 8, 10 and 14 (Fig. 3C,D,F), erythrocyte Se continued to accumulate in macaque no. 9 even after L-SeMet intubation ceased (Fig. 3E). When the dose was reduced from 300 to 25/~g Se/kg body weight/day for macaque no. 14, erythrocyte Se remained close to Cmax for the next 20 days (Fig. 3F) and plasma Se declined rapidly after dose reduction. Plasma concentrations greater than or equal to 2ppm in macaque no. 14 were associated with reduced body weight and poor appetite; Se did not accumulate in erythrocytes after cessation of L-SeMet treatment as seen in macaques nos 8 and 9 (Fig. 3C,E). As with macaque no. 9 (Fig. 3E), erythrocyte Se in macaque no. 15 (Fig. 3G) continued to increase even when the dose was reduced. With continuous oral L-SeMet exposure, a steady increase was observed in erythrocyte GPx (Fig. 2C) but not in plasma GPx (Fig. 2D). Although erythrocyte GPx tended to decline following discontinuation of L-SeMet intubation, the rise in erythrocyte GPx persisted and was equivalent to more than twice the pretreatment specific activity for at least 13 days after cessation of treatment with 300pg Se/kg body weight/day (Fig. 2C).
908
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Days Days Fig. 2. (A) Curves of mean erythrocyte Se concentration against time for female macaques given 30 consecutive daily oral doses of distilled, deionized water (O, three macaques), 25 ( 0 , two macaques), 150 (A, three macaques) or 300 (A, four macaques)/ag Se/kg body weight as L-SeMet. Values represent means + SE. (B) Curves of mean plasma Se concentration against time for female macaques given 30 consecutive daily oral doses of distilled, deionized water (O, three macaques), 25 ( 0 , two macaques), 150 (A, three macaques) or 300 (A, four macaques) /~g Se/kg body weight as L-SeMet. Values represent means _+ SE. (C) Curves of mean erythrocyte glutathione peroxidase (GPx) activity against time for female macaques given 30 consecutive daily oral doses of distilled, deionized water (O, three macaques), 25 (O, two macaques), 150 (A, three macaques) or 300 (A, four macaques) #g Se/kg body weight as L-SeMet. Values represent means _ SE. (D) Curves of mean plasma glutathione peroxidase (GPx) activity against time for female macaques given 30 consecutive daily oral doses of distilled, deionized water (O, two macaques), 25 ( 0 , two macaques), 150 (/k, three macaques) or 300 (A, four macaques) pg Se/kg body weight as L-SeMet. Values represent the means _+ SE. The m a x i m u m rates of urinary Se excretion occurred within the first 3-12 hr after dosing irrespective of the duration of exposure. The rate of urinary Se elimination between 12-24 hr remained constant after 15 daily doses of 25 pg Se/kg but increased by 59% relative to day 1 after 30 days of treatment. This rate was 57% greater after 15 consecutive daily doses
of 1 5 0 # g Se/kg (Fig. 1, Day 15) than after the first treatment with the same dose (Fig. l, Day 1). This increased rate of urinary Se excretion was also found after 15 consecutive daily doses of 3 0 0 ~ g Se/kg or 30 consecutive daily doses of 150 or 3 0 0 # g Se/kg (Fig. I, Day 30). The rate of urinary Se elimination between 12-24 hr on days 15 and 30 was greater in
Table 3. Plasma kinetic parameters of selenium following administration of multiple oral doses of L-selenomethionine (L-SeMet) to macaques TWA Dose (/ag L-SeMet/kg body weight/day)
25 25 62 117 150 150 150 188 190 203 300 300 300 300
Parameters* Animal no.
AUC ( # g . hr/ml)
17 16 15 14 13 11 12 9 10 8 3 7 4 6
184 449 873 1195 1643 2048 1800 3630 2203 2904 4645 5269 1958 3787
t ,2e (hr) 596 509 253 342 285 715 301 276 180 299 145 442 251 194
*Parameters are as defined in Materials and Methods. Values are for individual macaques numbered as in Table I.
Cma~(pg/ml) 0.45 0.87 2.17 3.35 2.37 3.02 3.15 4.51 4.13 5.20 7.26 8.19 3.03 5.73
Selenium pharmacokinetics
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