Circadian influence on effect of propranolol on exercise-induced tachycardia in healthy subjects

Summary Following a cross-over design propranolol 20 mg p.o. was given to 7 healthy subjects at 09.00 h and 21.00 h at an interval of 1 week. Heart rate (HR) during submaximal ergometer exercise was measured at four intervals during 10 h after treatment. Plasma propranolol concentrations were also determined.The suppressive effect (%R) of propranolol on the rise in HR during exercise after the morning dosage was significantly greater at 1.5 h and tended to be greater 3 h after administration than at comparable times in the evening trial. Mean plasma propranolol concentrations during the early phase were higher after the morning than the evening dose. The maximum plasma concentration (C_max), area under the plasma concentration-time curve from 0 to 10 h (AUC (0–10)) and absorption rate constant (k_a) were significantly greater after the morning dose. The time to maximum concentration (t_max) and elimination half-life (t_1/2) of the morning and evening dosages did not differ. A significant correlation was observed between plasma propranolol concentration and %R in HR during exercise in the morning (r=0.74) and evening (r=0.63) trials, and the regression lines of the morning and evening treatments did not differ.The data indicate that the suppressive effect of propranolol on exercise-induced tachycardia was relatively greater after a morning than an evening dose; that propranolol was more rapidly absorbed from the gastrointestinal tract after the morning than the evening dosage; that diurnal changes in the activity of propranolol depend in part on the time of administration and its subsequent effect on plasma concentrations of the drug; and that the antagonist activity of propranolol relative to a given drug concentration may not differ between morning and evening treatments.     

Chronopharmacokinetic studies of pranoprofen and procainamide

There is increasing evidence demonstrating that plasma drug concentrations are affected by their time of administration. In the current study, the chronopharmacokinetic profiles of an antipyretic agent, pranoprofen, and an antiarrhythmic agent, procainamide, were examined. In the first study, 75 mg of pranoprofen was given orally in seven healthy subjects at 10:00 (morning trial) or 22:00 (evening trial). In the second study, 500 mg of procainamide was given orally in eight subjects with premature ventricular contractions at 10:00 or 22:00. Blood samples for plasma drug concentrations were taken for a 10-hour (pranoprofen study) or a 24-hour (procainamide study) post-drug period. In the first (pranoprofen) study, the mean time to maximum concentration was significantly shorter, and the mean maximum plasma concentration as well as absorption rate constant had a tendency to be greater after the morning than after the evening trial. The mean area under the plasma concentration-time curve, elimination half-life or oral clearance of the morning and evening dosages did not differ. In the second (procainamide) study, no significant difference was observed in any pharmacokinetic parameter concerning procainamide or its active metabolite, N-acetyl-procainamide (NAPA) between the morning and evening trials. These data indicate that plasma levels of pranoprofen are affected by its administration time while plasma concentrations of procainamide and NAPA do not vary with the time of dosage.     

Chronopharmacological study of nitrendipine in healthy subjects

Nitrendipine 20 mg or placebo was given orally to eight healthy subjects in a cross-over design separated by 1 or 2 weeks. Drug was given at 9:00 AM (morning dosage) or at 9:00 PM (evening dosage). Systolic and diastolic blood pressure (SBP, DBP) were measured just before and 1, 2, 3, 4, 5, 7, 9, 12 and 24 hrs after treatment. Plasma nitrendipine concentrations were determined at 0.5, 1, 2, 3, 4, 5, 7, 9, 12 and 24 hrs and plasma catecholamines were measured at 2 and 5 hrs following drug administration. SBP did not decrease significantly after nitrendipine compared to after placebo at 9:00 AM or at 9:00 PM. DBP decreased significantly at 2, 3, 4 and 5 hrs after nitrendipine at 9:00 AM, but only at 4 hours after the 9:00 PM dose. Mean plasma nitrendipine concentrations during the absorption phase were lower after the evening dosage compared to the morning interval. Maximum plasma concentration (Cmax) was significantly lower and time to maximum concentration (tmax) tended to be longer after the evening dosage. Area under the plasma concentration-time curve from 0 to 24 hours (AUC0-24) and half-life of the terminal elimination phase (t1/2 beta) of the morning and evening dosages did not differ. A significant correlation was observed between plasma nitrendipine concentrations and changes in DBP during the drug treatment. Plasma noradrenaline concentrations were significantly higher 5 hours after nitrendipine compared to after placebo at 9:00 AM, but not at 9:00 PM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of dosing time on the pharmacokinetics and pharmacodynamics of a 'once-a-day' sustained release theophylline preparation.

1. The pharmacokinetics and the bronchodilating effect of theophylline were studied during 1 week of morning dosing and 1 week of evening dosing in a randomized cross-over design in thirteen patients with reversible airways obstruction, treated with a new 'once-a-day' theophylline capsule formulation. 2. There were no differences between the mean pre-dose trough plasma concentration, maximal plasma concentration, trough-to-peak variation, tmax, area under the plasma drug concentration time-curve after morning or evening dosage. There was only a slightly but significantly higher plasma theophylline concentration between 16 and 20 h after evening dosing. 3. The mean plasma drug concentration time-curves were relatively flat with a mean trough to peak variation of 73.9% after morning dosing and 66.7% after evening dosing. Both sets of mean data were within the 10-20 mg l-1 therapeutic range. 4. The variations in FEV1 and PEFR throughout the 24 h after either morning or evening dosing were similar and followed the normal pattern of diurnal variation. 5. Adverse effects were mild and occurred in three patients without causing discontinuation of the study. 6. The theophylline preparation used appears suitable for once a day administration either in the morning or in the evening.     

Time-dependent absorption of theophylline in man

Sixteen healthy volunteers were given either oral or intravenous doses of aminophylline (125 mg) at 9:00 A.M. and 9:00 P.M. under controlled food conditions. Measured at regular time intervals by homogeneous enzyme immunoassay, the plasma theophylline concentrations 1.5 and 2 hours after oral aminophylline were significantly higher in the morning than in the evening (P less than 0.05). Also, the mean peak plasma concentration was significantly higher (P less than 0.05) and the time to peak concentration was faster (P = 0.02) after the morning dose. Neither the morning mean elimination half-life nor the morning mean area under the plasma concentration-time curve differed significantly from those after the evening dose. After intravenous aminophylline, no significant differences were found in the plasma theophylline concentrations and in the elimination half-life between morning and evening. Therefore, the small but statistically significant time-dependent differences in theophylline kinetics must be due to changes in absorption from the gastrointestinal tract and not to changes in distribution or elimination of the drug.     

Chronotherapy of trichlormethiazide in hypertensive patients

Circadian influence of trichlormethiazide on serum electrolyte levels, lipid levels, and glucose levels were evaluated in 12 hypertensive patients. One tablet of trichlormethiazide (2 mg) was given once a day at 7:00 AM or 7:00 PM for 8 weeks. The study was done by a crossover design. Twenty-four-hour urine was collected, and fasting blood samples were obtained during the control period and at the end of each treatment period. The 24-hour urine level increased slightly after treatment with trichlormethiazide in the morning and evening trials. Urinary excretion of sodium also increased slightly in the morning trial and increased significantly in the evening trial. Serum concentrations of potassium and chloride decreased, and serum uric acid level increased after trichlor-methiazide treatment. No significant difference was observed in these parameters between morning and evening trials. Fasting blood glucose levels increased after trichlormethiazide treatment. The increment in blood glucose levels was greater in the evening trial than in the morning trial. These data indicate that the influence of trichlormethiazide on glucose tolerance might vary with its administration time.     

Time-Dependent Disposition of Tacrolimus and Its Effect on Endothelin-1 in Liver Allograft Recipients

Study Objective . To characterize a time-dependent disposition of oral tacrolimus and its relationship with plasma endothelin-1 concentrations. Design . Randomized, crossover study. Setting . Clinical research center of a university-affiliated hospital. Patients . Twelve stable liver transplant recipients. Interventions . In the steady state, 23 blood samples were taken from each patient before and after tacrolimus administration over 24 hours. Measurements and Main Results . Whole blood samples for tacrolimus and plasma endothelin-1 were analyzed by enzyme immunoassay. The relationship of their concentrations and their pharmacokinetic parameters between morning and evening doses were compared. The area under the curve (AUC) of tacrolimus in the morning dose was significantly larger than that in the evening dose (219 ± 54 ng•hr/ml and 188 ± 57 ng•hr/ml, respectively, p=0.004). The mean time to peak concentration (T_max) was significantly shorter for the morning dose than for the evening dose (1.6 ± 0.7 hrs and 3.5 ± 2.9 hrs, respectively, p=0.01). The mean peak concentration (C_max) was significantly higher in the morning dose than in the evening dose (32.2 ±9.1 ng/ml and 21.6 ± 8.3 ng/ml, respectively, p=0.008). However, the mean trough concentration (C_min) was not significantly different between doses. Endothelin-1 concentrations followed the same pattern as tacrolimus, with AUC and C_max for the morning significantly higher than those for the evening dose of tacrolimus (AUC 13.8 ± 3.7 pg•hr/ml, morning, and 11.0 ± 3.5 pg•hr/ml, evening, p=0.005; C_max 2.4 ± 1.1 pg/ml morning, and 1.5 ± 0.6 pg/ml evening, p=0.02). Tacrolimus levels did not correlate with endothelin-1 levels (r²=0.06, p=0.001). Conclusions . Tacrolimus disposition in liver transplant patients is determined by time of administration. Plasma endothelin-1 concentrations follow the same pattern as blood tacrolimus concentrations.     

Acute and chronic pharmacokinetics of asymmetrical doses of slow release choline theophyllinate in asthma.

The day and night pharmacokinetics of assymetrical doses of slow release choline theophyllinate (Sabidal SR 270) were compared at day 1 and at day 4 of treatment when steady state had been achieved. Ten patients with chronic asthma were given oral choline theophyllinate 424 mg at 09.00 h and 848 mg at 21.00 h for 4 days. At regular intervals during day 1 and day 4 of treatment theophylline concentrations were measured in plasma and dried blood spots by fluorimmunoassay. Theophylline concentrations measured from dried blood spots were slightly lower than those in plasma, the difference remaining constant at all time points during day 1 and day 4 of treatment. On day 1 the mean peak plasma theophylline concentration was 5.4 +/- 1.0 (+/- s.e. mean) micrograms ml-1 4 h after the morning dose and 11.2 +/- 1.6 micrograms ml-1 4 h after the evening dose which were significantly (P less than 0.01) different. Similarly the areas under the plasma theophylline concentration-time curves at night were significantly (P less than 0.001) greater than those observed during the day. During day 4 mean peak plasma concentrations of theophylline after the morning and larger evening dose were 13.2 +/- 1.3 and 12.1 +/- 1.4 micrograms ml-1 respectively, which were not significantly different. No significant difference was observed between the areas under the plasma theophylline concentration-time curves during the day and at night. However the post-dose time to peak was significantly delayed at night (6 h) compared to the morning (2 h, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Theophylline-terbutaline, a steady state study on possible pharmacokinetic interactions with special reference to chronopharmacokinetic aspects.

1. The pharmacokinetic interaction of terbutaline and theophylline and chronopharmacokinetics of both drugs were studied in a three-way crossover study with repeated administration of terbutaline (Bricanyl Depot) 7.5 mg twice daily, theophylline (Theo-Dur) 300 mg twice daily alone or the combination of both for 7 days to 12 healthy volunteers (six male and six female). 2. After the morning dose on day 7, blood and urine were sampled for 12 h, and after the evening dose on day 7, blood and urine were sampled for 48 h. Theophylline concentrations in plasma and concentrations of unchanged drug and metabolites in urine were determined by two selective high performance liquid chromatography methods. Terbutaline concentrations in plasma and urine were measured with a gas chromatography-mass spectrometry method. Area under the plasma concentration-time curve, fluctuations in plasma concentration, mean residence time, elimination half-life, renal clearance as well as maximal, minimal and average plasma concentration at steady state were evaluated. 3. The addition of terbutaline to the repeated administration of theophylline lowered the relative bioavailability of theophylline by approximately 11% during the night interval. The rate of elimination of theophylline and mean residence time were influenced accordingly. No significant changes in excretion of the theophylline metabolites were observed, but the excretion of 3-methylxanthine was slightly reduced by the concomitant terbutaline administration. None of the observed changes should be of any clinical importance. 4. The addition of theophylline did not influence any of the calculated pharmacokinetic parameters of terbutaline. 5. It can be concluded that no dosage adjustment is necessary when terbutaline and theophylline are given together.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of once-daily theophylline dose following the morning versus evening administration

The pharmacokinetics of theophylline (Uniphyllin) in blood and saliva was compared after morning and evening administration of a once-daily dose in a randomized cross-over study. Ten bronchial asthma patients received multiple doses of 800 mg theophylline at 8 a.m. and 8 p.m. under controlled food conditions. The precision of the serum concentration prediction from salivary measurements in individual patients was sufficient to obtain identical pharmacokinetic parameters and parallel concentration-time curves. There were no significant differences in the values of mean residence time and area under the concentration-time curves after morning and evening dosing, either in the kinetics of elimination and in the volume of distribution. The fluctuation of concentrations during the dosing intervals was 100% after morning doses and 80% after evening doses. No consistent interference with food was found. Circadian variation in the kinetics of drug disposition after once-daily theophylline administration did not occur. In order to achieve higher therapeutical levels at night and in the morning, the evening dosing seems to be preferable.     

Time-Dependent Absorption of Phenprobamate Following Multiple Dosing in Rats

Unusual serum profiles of phenprobamate, a centrally skeletal muscle relaxant, were observed in Sprague Dawley rats receiving multiple doses of phenprobamate suspension. The concentrations of phenprobamate were higher after the morning doses than after the evening doses, synchronizing with the day–night pattern of drug administration. Crossover studies were conducted to investigate the apparent time-dependent kinetics of phenprobamate. Phenprobamate emulsion was orally administered as a single dose to a group of six rats at 0900 hr and again, after a washout period of 3 days, at 2100 hr. Another group of six rats was treated similarly with intraperitoneal drug administration. Blood samples were collected at various times for 12 hr. The AUCs were 146.56 ± 31.77 µg · hr/ml for the morning oral dose and 111.31 ± 21.32 µg · hr/ml for the evening oral dose (P < 0.001). Administered intraperitoneally, the AUCs were 179.89 ± 37.50 and 185.58 ± 28.51 µg · hr/ml for the morning and evening doses, respectively, the difference of which was not significant. The paired t test indicated a significant morning–evening difference in AUC following oral but not intraperitoneal drug administration. This suggests the absorption rather than metabolism as a contributing factor to the time-dependent kinetics of phenprobamate in rats.     

Effects of the glucosidase inhibitor acarbose in patients with liver cirrhosis

In this preliminary study, we examined the effects of acarbose and placebo together with a standardized breakfast on blood glucose levels, on breath hydrogen excretion and on plasma insulin and glucagon levels; in addition, the effects on fasting blood levels of metabolites were studied following an evening meal with acarbose or placebo. Acarbose significantly reduced blood glucose levels in 10 patients with alcoholic cirrhosis following a meal containing 100 g of carbohydrate. There were no significant changes in plasma insulin after breakfast but glucagon levels were increased at 1 h after the meal. Breath hydrogen excretion did not change significantly. Acarbose given with a late evening snack reduced fasting beta-hydroxybutyrate levels the next morning in these cirrhotic patients.     

Clinical pharmacology of dilevalol (II). The pharmacokinetic, pharmacodynamic, and tolerance studies of dilevalol during repeated administration in healthy subjects

Dilevalol (50 mg) was given orally twice daily for eight days in six healthy subjects. All parameters were obtained following 1st (on day 1) and 15th (on day 8) dosages. Blood samples for plasma drug concentrations were taken for a 12-hour (after 1st dosage) or a 24-hour (after 15th dosage) post-drug period. Blood pressure (BP) as well as heart rate (HR) at supine position, during 50 degrees tilting and during a submaximal exercise were measured after each dosage. The mean time to maximum concentration (tmax) was faster, and the mean area under the plasma concentration-time curve (AUC) was greater after 15th dosage than following 1st dosage. No significant differences were observed in the maximum plasma concentration (Cmax), the distribution half-life (t 1/2 alpha) or the elimination half-life (t 1/2 beta) between the two dosages. BP at supine position as well as during 50 degrees tilting decreased significantly after each dosage, and did not differ between 1st and 15th dosages. Postural changes in BP or HR during 50 degrees tilting were not induced following 1st or 15th dosage. The suppressing effects (%R) on an increase in HR during a submaximal exercise were significantly larger after 15th dosage than after 1st dosage. A significant correlation was observed between plasma dilevalol concentration and %R in HR. These data indicate that the hypotensive effect of dilevalol is not altered during the repeated administration of the drug for 8 days. However, the beta-blocking activity of dilevalol might be enhanced during the repeated dosages, which is, in part, attributed to dosage-dependent elevation in plasma drug concentrations.     

Chronotherapeutic study of furosemide in hypertensive subjects: a preliminary report

In the present study, circadian influences of furosemide on serum electrolytes, lipids and glucose were evaluated in ten hypertensive subjects. A retard capsule (40 mg) of furosemide was given once a day in the morning (07 h 00) or in the evening (19 h 00) for eight weeks. The study was done through a cross-over design. Twenty-four hour urine was collected, and fasting blood samples were obtained during the control period and at the end of each treatment period. The 24-hour urine volume was slightly increased by the repeated administration of furosemide in the morning and evening trials. Urinary excretion of sodium also slightly increased in the morning trial and significantly increased in the evening trial. Serum concentrations of potassium and chloride decreased, while serum uric acid and triglyceride were increased by furosemide treatment. No significant difference was observed in these parameters between morning and evening trials. Fasting blood glucose increased following furosemide. The increment in this parameter was greater in the evening trial than in the morning trial. These findings indicated that the influence of furosemide on glucose tolerance might vary with its time of administration.     

Morning or evening dosage of omeprazole for gastro-oesophageal reflux disease?

Background and aims: When routinely checking patients receiving omeprazole treatment for gastro-oesophageal reflux, we have been finding patients with surprisingly low nocturnal gastric pH. The aim of this study was to evaluate the impact of timing of the 40 mg omeprazole once daily regimen. Methods: We evaluated the difference in effect of 40 mg omeprazole, given as a morning or evening dose, in 17 patients with gastro-oesophageal reflux disease. Gastric and oesophageal pH was recorded by portable 24-h two-channel pH-metry in a cross-over design of 14 days of morning and 14 days of evening administration. Results: In five patients pathological reflux was abolished by both regimens, four only during morning dosage, and three only during evening dosage. In the remaining five patients abolition of pathological reflux was not achieved. The therapeutic outcome and patient preference for morning or evening administration were closely related to the individual oesophageal pH curves. Patients with reflux induced by physical activity had a clear preference for morning dosage, patients with nocturnal reflux showed a clear preference for evening dosage. Gastric pH profiles showed a high inter-individual variation; paired statistics, however, revealed a significant impact of dosage timing on the gastric pH profile. After morning dosage the work-day part (the first 7 h) of the gastric pH profile is 0.72 ± 0.91 (mean difference of pairs ± s.d.) higher than after evening dosage (P < 0.01). After evening dosage the gastric pH during the supine period is 0.64 ± 0.83 (mean difference of pairs ± s.d.) higher than after morning dosage (P= 0.02). Conclusion: The timing of a 40 mg omeprazole dosage regimen has a clinically significant impact on the 24-h pH profile, and that—by relating to the patient 24-hour oesophageal pH-metry in combination with the patient symptomatology—the timing of this dosage is highly important for therapeutic efficacy.     

早、晚服用施慧达对原发性高血压疗效比较

目的评价早、晚服用施慧达对原发性高血压的降压疗效及毒副反应。方法将原发性高血压患者68例随机分为两组（分别命名为晨组与晚组），按2．5～5．0mg／d剂量分别于早餐或晚餐0．5h后口服施慧达4周，观察治疗前后降压疗效。结果与治疗前相比，两组对原发性高血压均具有显著降压效果，总有效率分别为85％、86％，但组间比较无统计学差异，两组中均未见有不良事件的发生。结论早、晚服用施慧达对原发性高血压均具有良好的降压效果，且均不易发生不良事件。     

Clinical chronopharmacology of oral sustained-release isosorbide-5-mononitrate in healthy subjects

Summary In 10 healthy male subjects the pharmacokinetics and haemodynamic effects of sustained-release isosorbide-5-mononitrate 60 mg (IS-5-MN) were studied after oral administration at two different times in the day (08.00 h and 20.00 h). Effects on blood pressure and heart rate after 3 min standing upright were measured in relation to the individual circadian control values.The pharmacokinetic parameters (C_max, t_max, AUC, t_1/2) did not differ after morning and after evening dosing, t_max being 5.2 h and 4.9 h, respectively. In contrast, the cardiovascular effects of IS-5-MN were clearly circadian phase-dependent. The maximum decrease in blood pressure decrease and increase in heart rate occurred significantly earlier after the evening (BPsys 2.8 h; BPdia 2.9 h; HR 3.8 h) than after the morning dose (BPsys 5.0 h; BPdia 6.0 h; HR 5.2 h). Thus, the peak haemodynamic effects coincided with the peak drug concentration after the morning dose, whereas the peak effect was in advance of the peak drug concentration after the evening dose of IS-5-MN.The data provide evidence of circadian phase-dependency in the dose-response relationship of oral IS-5-MN.     

Chronopharmacokinetic study of valproic acid in man: comparison of oral and rectal administration

Circadian stage-dependent changes of valproic acid (VPA) kinetics was examined after rectal administration comparing after oral dosing. Eight healthy volunteers took a single oral or rectal dose of VPA 400 mg, in a form of sodium valproate, on two occasions, at 8:30 A.M. or 8:30 P.M. Subjects were synchronized with diurnal activity and nocturnal rest as their usual life under standardized meal conditions. After oral administration, mean total VPA concentrations in plasma were significantly higher in the morning than in the evening during the absorption phase. Cmax tended to be higher, tmax was shorter (P less than 0.05) and absorption rate constant (ka) tended to be larger (0.05 less than P less than 0.1) for VPA in the morning than in the evening, although no difference was demonstrated in other pharmacokinetic values between morning and evening trials. After rectal administration, no significant difference was demonstrated in VPA kinetics between morning and evening trials. The rectal administration might have an advantage to eliminate the time-dependent changes of VPA kinetics. Thus the current practice of giving a rectal dose seems to be justified because the time-dependent changes in VPA kinetics observed after oral administration may be reduced without compromising therapeutic efficacy.     

Pharmacokinetics of a novel diltiazem HCl extended-release tablet formulation for evening administration

A novel diltiazem HCl extended-release (ER) tablet formulation was developed for evening administration in the management of angina and hypertension. Pharmacokinetics of the formulation were evaluated to identify variations in morning (7 a.m. or 8 a.m.) versus evening (10 p.m.) drug administration. Single-dose (360 mg) and multiple-dose (360 mg once daily for 7 days), open-label, randomized, two-way crossover studies of the new diltiazem HCl ER tablets were completed in 48 healthy volunteers. Serial plasma samples were collected via direct venipuncture up to 48 hours postdose and analyzed for diltiazem and its two major metabolites by high-performance liquid chromatography (HPLC). The primary parameters used to assess the data were AUC0- infinity, AUC0-tau, AUC6 a.m.-12 p.m., Cmax, and tmax. Statistical comparisons using ANOVA were evaluated after logarithmic transformation of dose-dependent parameters. Diltiazem HCl ER tablets administered in the evening exhibited 17% and 22% greater bioavailability compared to morning administration under single-dose and steady-state conditions, respectively. The two times of drug administration were bioinequivalent in both studies. The evening schedule also provided more than twofold higher plasma diltiazem levels in the critical morning hours, when both blood pressure and the incidence of cardiovascular events are the highest.     

Circadian variation in steady-state trough theophylline concentrations

The temporal variation in trough (i.e., predose) theophylline concentrations at steady state was examined. In general, adult subjects dosed with solid theophylline formulations have morning trough theophylline concentrations 10-16% greater than corresponding evening troughs; this morning-to-evening drop is statistically significant. Large-percentage diurnal changes (greater than +/- 20% and even greater than or equal to +/- 40%) in trough-to-trough serum theophylline concentrations are not uncommon in adults. However, the change in absolute trough theophylline concentrations is pronounced in only some individuals. Repetitive studies in four of five individuals tested generally demonstrated within-individual reproducibility in the direction (but not magnitude) of the temporal variation in trough theophylline concentration. This finding further substantiates the morning-to-evening change in trough theophylline concentration as a real phenomenon and not merely the result of random variability. It is believed that the temporal variability in trough concentration observed after peroral theophylline administration requires monitoring theophylline dosage needs by obtaining blood samples at the same time each day.