Drug metabolism: in vitro biotransformation of anabolic steroids in canines

Forensic drug testing of anabolic steroids in racing animals is required because of the potential for steroid abuse. Often when the metabolic products of an administered compound have not been identified, the analysis and verification of the administered compound is difficult. The objective of this study was to qualitatively identify the in vitro phase I biotransformation products of anabolic steroids that have a high potential for abuse in canines. The investigated steroids included testosterone, methyltestosterone, mibolerone and boldenone. Steroid biotransformation products were generated using beagle liver microsomes and analysed by high performance liquid chromatography (HPLC)/mass spectrometry (MS) with an electrospray ionization source. Characterization of steroid metabolites was based on HPLC retention, UV and mass spectra. The major testosterone metabolites were identified as androstenedione and 6beta- and 16alpha-hydroxytestosterone. 6beta-Hydroxymethyltestosterone was identified as a major metabolite in the methyltestosterone microsomal incubations. Several mibolerone metabolites were identified as monohydroxylated mibolerones as well as an oxidized mibolerone metabolite. Boldenone metabolites were identified as monohydroxylated boldenones, oxidized boldenone, and testosterone. This information should assist in the determination of anabolic steroid use in canines through the correlation of the urinary metabolites to the administered drug.     

Metabolism of Methenolone Acetate in a Veal Calf

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, α-methenolone was apparently the main one, but 1-methyl-5α-androstan-3,17-diol and 3α-hydroxy-1-methyl-5α-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. α-Methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.     

Estimation of quantitative proteinuria in the dog, using the urine protein-to-creatinine ratio from a random, voided sample

The correlation between 24-hour urine protein excretion and the protein-to-creatinine ratio (U-P/C) from random, voided urine specimens was assessed in 16 healthy Beagles (9 to 11 months old) and in 14 dogs with suspected renal proteinuria. Initially, a voided urine specimen was obtained from each dog, and the U-P/C was determined. An attempt was not made to standardize the time of collection of the voided urine. Subsequently, each dog was placed in a metabolism cage, and 24-hour urine specimens were collected for quantitative protein analysis. The Coomassie blue technique was used to measure urine protein. The correlation between the U-P/C and the 24-hour urine protein excretion (mg/kg/24 hr), evaluated by linear-regression analysis, was found to be significant (r = 0.975, P less than 0.01). These results substantiate previous findings and indicate that random, voided urine specimens may be used to compute the ratio and to accurately reflect 24-hour urinary protein loss in the dog.     

Effects of hydrochlorothiazide and diet in dogs with calcium oxalate urolithiasis

OBJECTIVE: To determine whether hydrochlorothiazide (HCTZ) reduces urinary calcium excretion in dogs with calcium oxalate urolithiasis. DESIGN: Original study. ANIMALS: 8 dogs with calcium oxalate urolithiasis. PROCEDURE: 4 treatment protocols were evaluated in each dog (a low calcium, low protein diet designed to prevent calcium oxalate urolith formation with and without administration of HCTZ [2 mg/kg (0.9 mg/lb) of body weight, PO, q 12 h] and a maintenance diet with higher quantities of protein and calcium with and without administration of HCTZ). At the end of each 2-week treatment period, 24-hour urine samples were collected. Blood samples were collected during the midpoint of each urine collection period. Analysis of variance was performed to evaluate the effects of HCTZ and diet on urine and serum analytes. RESULTS: Hydrochlorothiazide significantly decreased urine calcium and potassium concentration and excretion. Hydrochlorothiazide also significantly decreased serum potassium concentration. Compared with the maintenance diet, the urolith prevention diet significantly decreased urine calcium and oxalic acid concentration and excretion. Dogs consuming the urolith prevention diet had significantly lower serum concentrations of albumin and urea nitrogen. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of HCTZ decreased urine calcium excretion in dogs with a history of calcium oxalate urolith formation. The greatest reduction in urine calcium concentration and excretion was achieved when dogs received HCTZ and the urolith prevention diet. Results of this study suggest that the hypocalciuric effect of HCTZ will minimize recurrence of calcium oxalate urolith formation in dogs; however, long-term controlled clinical trials are needed to confirm the safety and effectiveness of HCTZ.     

Relationship of upper and lower urinary tract infection and bacterial invasion of uroepithelium to antibody-coated bacteria test results in female dogs

Urinary tract infection was demonstrated in 12 female dogs via bacteriologic culture of a specimen of bladder urine collected by antepubic cystocentesis. Escherichia coli was isolated in pure culture from the urine of 9 dogs. Urine specimens from 2 dogs contained E coli and alpha-streptococci and from 1 dog contained Streptococcus zymogenes in pure culture. In 6 dogs, urinary tract infection was limited to the urinary bladder, whereas 6 dogs had unilateral or bilateral culture-positive renal pelvic urine as well (specimens collected by percutaneous nephropyelostomy). An antibody-coated bacteria (ACB) test was conducted on a portion of the bladder urine specimen from each dog, and the urinary tissues from these 12 dogs and from 6 healthy, noninfected female dogs were examined at necropsy. Tissues were given a subjective score based on the severity of the lesions seen microscopically. Histologic scores, bacterial cultural results, and ACB test results were examined for significance. A significant difference was found in the histologic scores between infected and noninfected dogs (P less than 0.025), but comparisons among histologic scores, cultural results, and ACB test results were not significant among infected dogs. The ACB test could neither be used to localize bacterial infection within the urinary tract nor could it be used to indicate the presence of bacterial invasion of the uroepithelium in dogs.     

Reliability of taurine concentrations measured in single urine samples obtained from dogs eight hours after eating

OBJECTIVE: To evaluate the reliability of taurine concentrations measured in a single urine sample obtained from dogs 8 hours after eating, compared with taurine concentrations measured in 24-hour urine samples. ANIMALS: 18 healthy Beagles. PROCEDURE: After emptying the urinary bladder by transurethral catheterization, dogs were fed a canned maintenance diet. Approximately 8 hours later, urine, plasma, and serum samples were obtained for determination of fractional urinary excretion of taurine and urine taurine-to-creatinine concentration ratios (Utaur:Ucr). Results were compared with 24-hour urinary taurine excretion rate. RESULTS: Unbound and total fractional urinary taurine excretion correlated well with unbound and total 24-hour urinary taurine excretion. However, bound fractional urinary taurine excretion correlated poorly with bound 24-hour urinary taurine excretion. Unbound and total Utaur:Ucr correlated well with unbound and total 24-hour urinary taurine excretion. However, bound Utaur:Ucr correlated poorly with bound 24-hour urinary taurine excretion. CONCLUSION AND CLINICAL RELEVANCE: Fractional urinary excretion of unbound and total taurine, and unbound and total Utaur:Ucr are reliable indicators of 24-hour urinary unbound and total taurine excretion in healthy dogs. However, determination of 24-hour urinary taurine excretion is recommended for evaluating urinary bound taurine concentrations of dogs.     

Measurement of aldosterone in feline, canine and human urine

BACKGROUND: Systemic hypertension is an important problem in older cats associated with kidney disease and hypokalaemia, suggesting that excessive activity of the renin-angiotensin-aldosterone system might contribute to the hypertensive state. Fluctuations in plasma renin activity and plasma aldosterone concentrations complicate the interpretation of these assays. OBJECTIVES: The aim of this study was to determine whether measurement of urinary aldosterone excretion in cats aided the investigation of hypertension. METHODS: Urine concentrations of free (ethyl acetate extract) and 18-glucuronidated aldosterone (acid hydrolysis before extraction) were measured by radioimmunoassay in normal, normotensive and hypertensive azotaemic cats (n=11 per group). Urine samples from 11 healthy human volunteers and eight normal dogs were also analysed for comparison. Urinary aldosterone concentration was corrected for the urinary creatinine concentration. RESULTS: Cats excreted 7.3 times less free aldosterone than human beings, and no free aldosterone was detected in dog urine. Acid hydrolysis led to large increases in aldosterone recovery from both human beings and dog but not feline urine. No significant effect of hypertension or azotaemia on feline urinary aldosterone concentration was found. CLINICAL SIGNIFICANCE: Measurement of aldosterone in feline urine using the available methodology has limited or no utility in investigating feline hypertension.     

Urine metabolite values in fed and nonfed clinically normal beagles.

Twenty-four-hour excretion of urine metabolites was determined in 33 clinically normal Beagles during periods of consumption of a standard diet and when food was withheld. The goal was to determine normal canine values for urine analytes incriminated in the genesis of calcium oxalate uroliths. During periods when dogs consumed food, dairy urinary excretion of calcium, uric acid, sodium, potassium, magnesium, ammonium, and hydrogen ions were significantly (P = 0.0004, 0.0038, 0.001, 0.0001, 0.0004, 0.0001, and 0.024, respectively) higher than when food was withheld. Urinary excretion of phosphorus, oxalate, and citrate were not significantly different between samples obtained during periods of food consumption and when food was withheld. Male dogs excreted significantly higher quantities of urine oxalate than females during fed (P = 0.003) and nonfed (P = 0.003) conditions. When food was withheld, urinary uric acid excretion was significantly higher in males than females (P = 0.01). Females excreted significantly more urine calcium than males when food was withheld (P = 0.003). Our results indicated that dietary conditions influence the quantity of sodium, potassium, calcium, magnesium, and uric acid excreted in the urine of clinically normal dogs; therefore, dietary conditions should be considered when measuring the concentration of these analytes in urine.     

Absorption, distribution, metabolism, and excretion of dirlotapide in the dog

Three once-daily oral doses of 0.2 mg/kg [¹⁴C]dirlotapide were administered to beagle dogs to study the absorption, distribution, metabolism, and excretion of dirlotapide. Mean ¹⁴C recovered at 2.5 and 4.5 h after the last dose was 90%. Mean ¹⁴C in urine, bile, and feces was <1%, 1.7%, and 56% of the dose, respectively. In tissues, 26% of the ¹⁴C dose was present in the gastrointestinal tract, 6.0% in liver, and <1% each in kidney, gall bladder, heart, and brain. To further characterize drug disposition, a single 2.5-mg/kg oral dose of [¹⁴C]dirlotapide was administered to beagle dogs. More than 84% of the dose had been eliminated by 72 h in feces, with 21% of the dose present in feces as parent dirlotapide. Less than 1% of the dose was excreted in urine. In bile collected during the first 24-h postdose from three dogs, 32% and 11% of the ¹⁴C dose was present in samples from male and female dogs, respectively. Based upon metabolite profiling of plasma, excreta, and bile samples, dirlotapide was extensively metabolized to more than 20 metabolites. Biliary/fecal excretion and the potential for enterohepatic recycling of metabolites are suggested.     

Metabolism of endogenous and exogenous anabolic agents in cattle

The endogenous anabolic agents, estradiol-17 beta, progesterone and testosterone are steroids that are quickly metabolized by the liver and are not very active when administered orally. Estradiol-17 beta is excreted by the bovine in bile as free estradiol-17 alpha, and by swine, in urine, as glucuronides and sulfates. In ruminants, the primary metabolite of progesterone is pregnandiol and that of testosterone is epitestosterone. In horses, the metabolites of these compounds are primarily 17 ketosteroids. Esters of the endogenous anabolic agents are rapidly hydrolyzed and the nonesterified forms follow the same biotransformation pathways as the natural compounds biosynthesized by the animal. The exogenous anabolic agents, such as trenbolone acetate, zeranol and diethylstilbestrol, may be active by the oral route and are less readily metabolized in the liver than the endogenous anabolics. Their metabolic pathways may be complex and lead to excreted forms after glucuronide conjugation. With respect to biochemical mechanism of action, it can be assumed that the anabolics act like all steroids by way of intracellular receptors. Biotransformations could lead to more reactive molecules that may bind themselves to normal constituents of the organism. Bound metabolites are generally formed later than free metabolites, and are considered less toxic with a lower level of bioavailability.     

Estimation of Quantitative Enzymuria in Dogs With Gentamicin-Induced Nephrotoxicosis Using Urine Enzyme/Creatinine Ratios From Spot Urine Samples

The correlation between 24-hour urinary excretion of N -acetyl-β- d -glucosaminidase (NAG) and γ-glutamyl transferase (GGT) with urine NAG and GGT/creatinine ratios was assessed in dogs with gentamicin-induced nephrotoxicosis. Eighteen 6-month-oid male Beagles with normal renal function were randomly divided into 3 groups of 6. Each group was fed a different concentration of protein (high protein, 27.3%; medium protein, 13.7%; and low protein, 9.4%) for 21 days. After dietary conditioning, gentamicin was administered at a dose of 10 mg/kg IM tid for 8 days and each group was continued on its respective diet. Endogenous creatinine clearance and 24-hour urinary excretion of NAG and GGT were determined after dietary conditioning (day 0) and on days 2, 4, 6, and 8 of gentamicin administration. In addition, urine NAG and GGT/creatinine ratios (IU/L ± mg/dL) were determined from catheterized spot urine samples obtained between 7 and 10 am on the same days. The correlation between 24-hour urinary enzyme excretion and urine enzyme/creatinine ratio in the spot urine samples was evaluated by simple linear regression analysis. Spot sample urine enzyme/creatinine ratios were significantly correlated with 24-hour urinary enzyme excretion through day 4 for dogs on low dietary protein, through day 6 for those on medium protein, and through day 8 for those on high dietary protein. Mean ± SD baseline values for urine NAG/creatinine ratio and 24-hour urinary NAG excretion were 0.06 ± 0.04 and 0.19 ± 0.14 IU/kg/24 hr, respectively. Baseline values for urine GGT/creatinine ratio and 24-hour urinary GGT excretion were 0.39 ± 0.18 and 1.42 ± 0.82 IU/kg/24 hr, respectively.     

Use of protein-to-creatinine ratio in a single urine specimen for quantitative estimation of canine proteinuria

The daily excretion of urinary protein was evaluated in 8 conditioned research dogs and in 10 hospitalized, proteinuric dogs, using 24-hour urine collections. Concurrent with each 24-hour urine collection, a 5- to 10-ml urine specimen was obtained during midday. The ratio of urine protein to urine creatinine concentration was determined from the single urine specimen for each dog. Linear regression analysis was used to calculate the correlation between that ratio and the 24-hour urinary protein loss (mg/kg of body weight). The coefficient of determination was significant (r2 = 0.95, P less than 0.0001). Determination of the protein-to-creatinine ratio in a single urine specimen was found to be a sensitive, rapid, and dependable diagnostic technique for detection and quantitative estimation of proteinuria.     

Pharmacokinetics of sulfamethazine in male, female and castrated male swine

The concentration of sulfamethazine in plasma and sulfamethazine and its metabolites in urine were compared in male, female and castrated male swine. A surgical technique for placement of catheters in the urinary bladder was used to facilitate the collection of urine in males and castrated males. The elimination rate of sulfamethazine from plasma and the excretion of parent drug and metabolites into urine did not differ significantly among females, males and castrated male swine.     

Effect of time of cisplatin administration on its toxicity and pharmacokinetics in dogs.

Cisplatin (90 mg/m2) was administered in a 5-minute bolus IV infusion to dogs at 8 AM (n = 6) or 4 PM (n = 6). Blood and urine samples were collected over a 4-hour period for statistical moment pharmacokinetic analysis. Mean urinary excretion rate of total platinum was increased, whereas mean plasma residence time of ultrafilterable platinum was decreased, in the group treated at 4 PM (PM group), compared with those treated at 8 AM (AM group). Over a 2-week postinfusion-monitoring period, both groups of dogs developed decreases in creatinine clearance, urine/serum osmolality ratio (UOsm/SOsm), specific gravity, and increase in BUN, serum creatinine concentration, urine gamma-glutamyltranspeptidase/urine creatinine ratio (UGGT/UCr), fractional excretion of magnesium, and fractional excretion of phosphate. Urine specific gravity and UOsm/SOsm were significantly decreased, whereas UGGT/UCr and BUN were significantly increased in the AM group, compared with the PM group. The time of administration had a significant effect on the pharmacokinetics of cisplatin, which resulted in significant differences in cisplatin-induced renal toxicosis.     

Comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis

OBJECTIVES: To identify an appropriate sampling technique(s) to accurately detect the bacteria causing urinary tract infections in dogs with urolithiasis. METHODS: Twenty-one dogs with urolithiasis were included in the study. Three types of samples were taken from each dog. Urine was collected by cystocentesis, and a urinary bladder mucosal biopsy and urolith were retrieved during cystotomy. The samples were then cultured on blood agar and MacConkey's agar to identify the bacteria associated with urinary tract infections. RESULTS: Bacterial urinary tract infection was found in 16 cases (76.19 per cent). The most prevalent bacteria found to cause urinary tract infection were Escherichia coli (n=7), followed by coagulase-positive Staphylococcus species (n=4), Klebsiella pneumoniae (n=2), Pseudomonas aeruginosa (n=2) and Proteus mirabilis (n=1). In the case of a positive urine culture, the same bacteria were also cultured from the urinary bladder mucosal biopsy alone or from both the urinary bladder mucosal biopsy and urolith. However, in the case of a negative urine culture, bacteria were found to be present in the urinary bladder mucosal biopsy or urolith cultures in 23.81 per cent of dogs. The uroliths that gave positive culture results were either infection-induced uroliths composed of struvite and calcium carbonate phosphate, ammonium acid urate only or metabolic uroliths composed of calcium oxalate and calcium phosphate, or calcium phosphate only. All the uroliths that gave negative culture results were metabolic uroliths composed of calcium oxalate and/or calcium phosphate, and uric acid and calcium phosphate. CLINICAL SIGNIFICANCE: When the culture from the urine obtained by cystocentesis is negative, cultures of urinary bladder mucosal biopsy and urolith are recommended in dogs with urolithiasis in order to accurately assess the microbiological status of the urinary tract.     

Effects of dietary supplementation with sodium chloride on urinary relative supersaturation with calcium oxalate in healthy dogs

OBJECTIVE: To evaluate the effect of dietary supplementation with sodium chloride (NaCl) on urinary calcium excretion, urine calcium concentration, and urinary relative supersaturation (RSS) with calcium oxalate (CaOx). ANIMALS: 6 adult female healthy Beagles. PROCEDURE: By use of a crossover study design, a canned diet designed to decrease CaOx urolith recurrence with and without supplemental NaCl (i.e., 1.2% and 0.24% sodium on a dry-matter basis, respectively) was fed to dogs for 6 weeks. Every 14 days, 24-hour urine samples were collected. Concentrations of lithogenic substances and urine pH were used to calculate values of urinary RSS with CaOx. RESULTS: When dogs consumed a diet supplemented with NaCl, 24-hour urine volume and 24-hour urine calcium excretion increased. Dietary supplementation with NaCl was not associated with a change in urine calcium concentration. However, urine oxalate acid concentrations and values of urinary RSS with CaOx were significantly lower after feeding the NaCI-supplemented diet for 28 days. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary supplementation with NaCl in a urolith-prevention diet decreased the propensity for CaOx crystallization in the urine of healthy adult Beagles. However, until long-term studies evaluating the efficacy and safety of dietary supplementation with NaCl in dogs with CaOx urolithiasis are preformed, we suggest that dietary supplementation with NaCl be used cautiously.     

Urinary excretion of pentoxifylline and its metabolites by standardbred mares.

The urinary excretion of a sustained-release formulation of pentoxifylline was studied in the horse after the oral administration of 4.0 grams of Trental tablets. Urine samples were collected for 24 hours after dosing and analyzed for pentoxifylline and its metabolites using high-performance liquid chromatography coupled with an ultraviolet detector. Six metabolites of pentoxifylline were identified in horse urine in addition to less than 0.2% of unchanged drug. Concomitant use of gas chromatography/mass spectrometry allowed for the elucidation of the chemical structures of the metabolites. Metabolism of pentoxifylline yields one demethylated derivative, four hydroxylated metabolites and a conjugate of one of the hydroxymetabolites as urine products. The demethylated derivative, 3-methyl-1-(5-oxohexyl)-xanthine, was found to be the predominant metabolite in the urine.     

Pharmacokinetics of oral amantadine in greyhound dogs

This study reports the pharmacokinetics of amantadine in greyhound dogs after oral administration. Five healthy greyhound dogs were used. A single oral dose of 100 mg amantadine hydrochloride (mean dose 2.8 mg/kg as amantadine hydrochloride) was administered to nonfasted subjects. Blood samples were collected at predetermined time points from 0 to 24 h after administration, and plasma concentrations of amantadine were measured by liquid chromatography with triple quadrupole mass spectrometry. Noncompartmental pharmacokinetic analyses were performed. Amantadine was well tolerated in all dogs with no adverse effects observed. The mean (range) amantadine C_MAX was 275 ng/mL (225–351 ng/mL) at 2.6 h (1–4 h) with a terminal half‐life of 4.96 h (4.11–6.59 h). The results of this study can be used to design dosages to assess multidose pharmacokinetics and dosages designed to achieve targeted concentrations in order to assess the clinical effects of amantadine in a variety of conditions including chronic pain. Further studies should also assess the pharmacokinetics of amantadine in other dog breeds or using population pharmacokinetics studies including multiple dog breeds to assess potential breed‐specific differences in the pharmacokinetics of amantadine in dogs.     

Comparison of the efficacy of two anti-uric acid diets in dalmatian dogs.

Commercial anti-uric acid diets for dogs may contain insufficient protein to sustain growth and lactation. In order to investigate the efficacy of an experimental purine-free diet moderately low in protein, its effect on urinary uric acid excretion was compared with that of a commercial dog food and a commercial low-protein anti-uric acid diet. The experimental diet, commercial dog food and commercial anti-uric acid diet contained 10.0, 12.8 and 5.0 g crude protein/MJ metabolizable energy, respectively. Twelve Dalmatian dogs were subjected to a 3 x 3 Latin square study. Although the plasma uric acid concentration was significantly lower when the dogs were fed either the commercial anti-uric diet (18.7 +/- 6.0 mumol/l, mean +/- SD, n = 12) or the experimental diet (19.2 +/- 8.3 mumol/l), when compared to the commercial dog food (29.2 +/- 11.1 mumol/l), no significant decrease of uric acid concentration in urine collected before the morning meal was seen. The average concentration of urinary uric acid was 60 mumol/l. There was a significant increase in the urea:creatinine ratio in urine when the dogs were fed the experimental diet compared to the commercial anti-uric acid diet, confirming that the experimental diet contained more protein. The experimental diet lowered plasma uric acid and was relatively high in protein and thus may be suitable for use in growing and lactating dogs to prevent ammonium urate urolithiasis.     

Free amino acids in the plasma and urine of dogs from birth to senescence.

Plasma and urinary amino acid concentrations were determined from a cross-sectional sample of dogs of all ages. Reference or base line values for these components were established for five different age groups. A general decrease in urinary excretion of amino acids for each gram of creatinine was found during the developmental period. This decrease was not correlated to changes in plasma concentrations, which varied for the individual amino acids. The decrease did appear to be related to the functional maturation of the kidney. Several methylated basic amino acids were identified in the urine. Of these, NG, NG- and NG,N'G-dimethylarginine, and trimethyllysine had not been previously reported as constituents of dig urine, but are major components of the urinary amino acids, expecially in newborn animals. The 3-methylhistidine is also excreted in large amounts by dogs and measurable quantities of this compound appear in the plasma. These findings suggest an active metabolic function for methylated amino acids in the dog.