Effects of Ketamine on Metabolomics of Serum and Urine in Cynomolgus Macaques (Macaca fascicularis).

In this study, a metabolomics approach based on nuclear magnetic resonance spectroscopy and pertinent multivariate data analyses was used to evaluate the effect of ketamine on metabolic markers in cynomolgus macaques. Principal component analysis and orthogonal projection to latent structure with discriminant analysis showed that ketamine (10 mg/kg) induced metabolic perturbations. Compared with the control group, ketamine-treated macaques had lower serum levels of α-glucose, myoinositol, lactate and succinate and lower urine levels of pyruvate and lactate. In contrast, the levels of leucine in serum and arginine in urine were significantly higher in the ketamine group. Our results also demonstrated that a single injection of ketamine influenced the major energy and amino acid metabolic pathways in cynomolgus macaques. Our study suggests that these influences should be considered in the design of experiments and the interpretation related blood and urine data from ketamine-sedated cynomolgus macaques.

Competitive inhibition of SGLT2 by tofogliflozin or phlorizin induces urinary glucose excretion through extending splay in cynomolgus monkeys.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.

Concentration compared with total urinary excretion of 11,17-DOA in cynomolgus monkey urine.

Strees sensitive molecules exhibit great variation in concentration in the circulation and it may often be advantageous to quantify these in urine or feces rather than in serum or plasma. We advocate that all urine-or feces-should be collected, and that excretion of stress sensitive molecules should be expressed as amounts excreted per time unit per kg body-weight, rather than being expressed as concentrations in samples. Urine and feces excretion varies significantly within and between animals over time, which may render simple concentration measures of molecules of little biological relevance.

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis).

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.

Effect of dioxin on ovarian function in the cynomolgus macaque (M. fascicularis).

Ovarian function was evaluated in mature female cynomolgus macaques 443 to 625 days following a single oral exposure (1, 2, or 4 microg/kg BW) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Urinary estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and follicle stimulating hormone (FSH) were measured. Three of four animals in the high dose group had no evidence of menstrual cycles while animals in the low and medium dose groups plus one from the high dose group had cycles that were similar to those of control animals. The noncycling animals had baseline E(1)C concentrations without ovulatory midcycle peaks and monotonic PdG profiles. Mean FSH concentrations during the midfollicular phase of the medium dose group and during the entire cycle of the high dose group were elevated compared to those of the control group and the endometria of the noncycling animals were inactive. These data demonstrate that a single exposure of 4 microg/kg BW TCDD leads to long-term adverse effects on ovarian function in primates.

Alkaptonuria in a cynomolgus monkey (Macaca fascicularis).

An eight year old wild caught Cynomolgus monkey was diagnosed as having alkaptonuria, a condition characterized by the passage of a normal-colored urine which darkens upon standing. The underlying cause is the congenital lack of homogentisic acid oxidase with subsequent passage of homogentisic acid in the urine. No other clinical manifestations, such as deposition of pigment in the skin or mucous membranes or development of an ochronitic arthritis, were observed in this animal.

Enzyme immunoassays for ovarian steroid metabolites in the urine of Macaca fascicularis.

In vivo studies using carbon 14 labeled estradiol (E2) and progesterone (Po) were performed to characterize the time course and metabolic fate of circulating E2 and Po. Co-chromatography of human, orangutan, and macaque luteal phase urine samples demonstrated the presence of a steroid conjugate peak in all three species that was identified as being androsterone and etiocholanolone glucuronides. An enzyme immunoassay for urinary metabolites of Po was developed subsequently for Macaca spp. using a monoclonal antibody that cross-reacted with both C-19 and C-21 metabolites.

The use of a urinary estrone conjugates assay for detection of optimal mating time in the cynomolgus macaque (Macaca fascicularis).

Forty-four female cynomolgus macaques (Macaca fascicularis) were examined to determine the optimum fertile period for mating. Daily urinary estrone conjugates (E1C) were measured, beginning on day 7 of the menstrual cycle, until a 1.5-gold E1C rise above the baseline was detected. The females were bred the next morning. Pregnancies were verified in all animals at day 18 postbreeding, and/or on day 25 postbreeding. Serum progesterone levels were used to correlate the relationship between ovulation and the E1C peak. Forty-four of the 57 cycles indicated a urinary E1C peak between days 10-15 of the menstrual cycle; this peak occurred on the day following the initial 1.5-fold to twofold rise in 90% of the cycles. A single 2-hr mating period the day before, the day of, or the day after the E1C peak resulted in conception in 17 of 44 (38.6%) animals.

Identification of a non-steroidal estrogen, equol, in the urine of pregnant macaques: correlation with steroidal estrogen excretion.

Macaque urinary estrogens at late pregnancy were separated by high performance liquid chromatography and quantified, both with radioimmunoassay and an in vitro uterine estrogen receptor assay. Five estrogens were measured. Four were steroids: estriol, estrone, 17 beta-estradiol, and 16 alpha-hydroxyestrone. The fifth was a flavonoid, equol, a metabolite of plant isoflavonoids, formononetin and genistein. By mass, estrone and equol were the predominant urinary estrogens, with equol reaching levels of microgram/mg creatinine in three of 8 pregnancies studied. Both quality and quantity of urinary estrogen excretion in the rhesus (Macaca mulatta) was compared to those in 4 other species (Macaca fascicularis, Macaca nemestrina, Macaca radiata and Macaca silenus). All 5 estrogens present in the rhesus were also present in the other 4. Variability in mass of each estrogen excreted appeared no greater between species than within the rhesus. In a longitudinal study, urinary equol levels were most highly correlated with those of estrone, the predominant excretory steroid of macaque pregnancy. We conclude endogenous steroidal estrogen is related to production of equol in macaques, however, equol is not dependent on the feto-placental unit as low levels of equol were also present in male macaque urine.

[Water intake and urinary output in rhesus (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis)].

Water intake (drinking water and water from food) and urinary output in rhesus (Macaca mulatta) and cynomolgus monkey (M. fascicularis) watered ad libitum in individual cage were examined with 52 animals. The animal room was maintained at 25 +/- 2 degrees C and 55 +/- 10% with relative humidity, being lit at 12 hours interval. Water intake was found in wide variation between individuals, and about 20% of the monkeys were recognized as polydipsia without another abnormal behavior. No significant variation was found between daily water intake, but periodical (or seasonal) variation was observed in polydipsic monkey. Urinary output changed accordingly with water intake. Rate of the urinary output to the water intake was 76% in male rhesus, 59% in male cynomolgus and 49% in female cynomolgus. The rate was higher in the polydipsia than in the normal monkey. Diurnal patterns of water drinking and urinary output indicated that the monkeys were mostly active during the hours of lighting. Some effects of experimental procedure were observed on drinking and voiding in cynomolgus monkeys while no effect on rhesus monkeys. Prandial drinking was observed in monkeys as was reported on other laboratory animals such as rats.

Metabolism of prostacyclin in Cynomolgus monkey.

The metabolism of prostacyclin (PGI2) in vivo was investigated in Cynomolgus monkey. Following intravenous infusion of 11-[3H]-PGI2 for three days, pooled urine was extracted with Amberlite XAD-2, then chromatographed and purified by Sephadex LH-20, and reverse phase column chromatography. Radioactive fractions were converted to appropriate derivatives for identification by gas chromatography mass spectrometry. Twelve metabolites were characterized, the major of which was 6-keto-PGF1 alpha, accounting for 13% of the urinary radioactivity. The metabolic pathways are similar to those observed earlier in the rat. The excretion of substantial amounts of unchanged 6-keto-PGF1 alpha indicated that the monkey was not able to metabolize PGI2 as avidly as the rat.