Global Metabolomic Identification of Long-Term Dose-Dependent Urinary Biomarkers in Nonhuman Primates Exposed to Ionizing Radiation.

Due to concerns surrounding potential large-scale radiological events, there is a need to determine robust radiation signatures for the rapid identification of exposed individuals, which can then be used to guide the development of compact field deployable instruments to assess individual dose. Metabolomics provides a technology to process easily accessible biofluids and determine rigorous quantitative radiation biomarkers with mass spectrometry (MS) platforms. While multiple studies have utilized murine models to determine radiation biomarkers, limited studies have profiled nonhuman primate (NHP) metabolic radiation signatures. In addition, these studies have concentrated on short-term biomarkers (i.e., <72 h). The current study addresses the need for biomarkers beyond 72 h using a NHP model. Urine samples were collected at 7 days postirradiation (2, 4, 6, 7 and 10 Gy) and processed with ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight (QTOF) MS, acquiring global metabolomic radiation signatures. Multivariate data analysis revealed clear separation between control and irradiated groups. Thirteen biomarkers exhibiting a dose response were validated with tandem MS. There was significantly higher excretion of l-carnitine, l-acetylcarnitine, xanthine and xanthosine in males versus females. Metabolites validated in this study suggest perturbation of several pathways including fatty acid ß oxidation, tryptophan metabolism, purine catabolism, taurine metabolism and steroid hormone biosynthesis. In this novel study we detected long-term biomarkers in a NHP model after exposure to radiation and demonstrate differences between sexes using UPLC-QTOF-MS-based metabolomics technology.

Early diet impacts infant rhesus gut microbiome, immunity, and metabolism.

Epidemiological research has indicated a relationship between infant formula feeding and increased risk of chronic diseases later in life including obesity, type-2 diabetes, and cardiovascular disease. The present study used an infant rhesus monkey model to compare the comprehensive metabolic implications of formula- and breast-feeding practices using NMR spectroscopy to characterize metabolite fingerprints from urine and serum, in combination with anthropometric measurements, fecal microbial profiling, and cytokine measurements. Here we show that formula-fed infants are larger than their breast-fed counterparts and have a different gut microbiome that includes higher levels of bacteria from the Ruminococcus genus and lower levels of bacteria from the Lactobacillus genus. In addition, formula-fed infants have higher serum insulin coupled with higher amino acid levels, while amino acid degradation products were higher in breast-fed infants. Increases in serum and urine galactose and urine galactitol were observed in the second month of life in formula-fed infants, along with higher levels of TNFα, IFN-γ, IL-1ß, IL-4, and other cytokines and growth factors at week 4. These results demonstrate that metabolic and gut microbiome development of formula-fed infants is different from breast-fed infants and that the choice of infant feeding may hold future health consequences.

Reference values of hematology, chemistry, electrolytes, blood gas, coagulation time, and urinalysis in the Chinese rhesus macaques (Macaca mulatta).

BACKGROUND: Due to increasing preclinical study using the rhesus monkeys, the physiological data of these monkeys are very important for the objective evaluation of experimental results. Here, we report the physiological values of the Chinese rhesus monkeys acclimated under a well-controlled laboratory environment. METHODS: Seventy healthy rhesus monkeys of both genders (29 males and 41 females) were used in this study. Available data on hematology, serum biochemistry, electrolytes, blood gas, coagulation time, urinalysis, water consumption, urine volume, and body weight were examined. RESULTS: The lymphocyte values were 1.5-2.5 times higher than the neutrophil values in both genders. In serum biochemistry, there was no significant difference between the two genders. Interestingly, the values of alkaline phosphatase from the monkeys were very high. According to isoenzyme analysis, the percentage of alkaline phosphatase that originated from the bone was 60%, and this value was significantly higher than that from the liver (39%, P = 0.020). CONCLUSION: Physiological data are very important parameters that can be directly and indirectly related to organ function (e.g. islet, kidney, liver, heart, etc.) in the transplantation model. In this respect, our data constitute valuable references for preclinical study using the Chinese rhesus monkeys.

Altered hypothalamic-pituitary-adrenocortical function in rhesus monkeys (Macaca mulatta) with self-injurious behavior.

Individually housed rhesus monkeys sometimes spontaneously develop self-injurious behavior (SIB) in the form of self-directed biting that, on occasion, results in severe tissue damage and mutilation. We previously demonstrated lower levels of plasma cortisol in rhesus monkeys with a history of self-wounding (SW) when compared to non-wounders (NW). Furthermore, cortisol levels were negatively correlated with rates of self-directed biting. The present study was designed to further characterize the relationships between hypothalamic-pituitary-adrenocortical (HPA) activity, self-wounding, and self-directed biting. Basal 24-h urinary free cortisol excretion, the urinary free cortisol response to a low dose of dexamethasone, and the plasma cortisol response to ACTH were examined in 24 individually housed rhesus monkeys, based on wounding history, i.e. the presence/absence of a veterinary record of self-wounding, and current rates of self-directed biting, i.e. the median split of self-directed biting frequency (independent of wounding status). There were no reliable group differences on any of the physiological measures when analyzed by wounding history. However, the plasma cortisol response 30 min post-ACTH stimulation was significantly correlated with wounding recency, such that lower responsivity was associated with more recent wounding episodes. When the results were analyzed on the basis of biting frequency, high frequency biters (HFB) compared to low frequency biters (LFB) showed decreased HPA negative feedback sensitivity to dexamethasone and a trend towards an attenuated plasma cortisol response to ACTH stimulation. These findings suggest that SIB in socially reared monkeys is associated with complex changes in HPA axis function that are related to the expression of the pathology, i.e. self-directed biting, and to the recency of a wounding episode. It remains to be determined whether humans who exhibit SIB show similar alterations in HPA function.

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 refractometer index as a correction factor for urinary estradiol in rhesus females.

Refractometer indexes were standardized and found to be highly correlated (r = 0.831) to actual creatinine levels. Urinary estradiol corrected by creatinine levels and refractometer indexes were found to be highly correlated (r = 0.857). Predicted ovulation was the same day in 86% of the ovulatory cycles predicted by creatinine and refractometer corrected estradiol levels. Refractometer indexes may be used in place of creatinine levels to correct for urine concentration fluctuations when predicting ovulation in the rhesus female.

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.

Adrenergic and dopaminergic response to chronic chair restraint in the rhesus monkey.

Prolonged chair restraint and social isolation in the rhesus monkey led to a reduction in the urinary excretion of HVA (4-hydroxy-3-methoxyphenylacetic acid), DOPAC (3,4-dihydroxyphenylacetic acid), VMA methoxy-4-hydroxymandelic acid), and MHPG (3-methoxy-4-hydroxyphenylethylglycol) over a 3 week period. This adaptation to a chronically "stressful" situation corresponds to earlier studies on the rhesus monkey indicating a gradual reduction in the urinary excretion of norepinephrine and epinephrine after initiation of restraint. The following basic information on the urinary excretion of catecholamine metabolites was obtained: (1) the rate of excretion of the dopamine metabolites (HVA and DOPAC) is about four times higher than the rate of excretion of adrenergic metabolites (VMA and MHPG): (2) MHPG is the major adrenergic metabolite in the rhesus monkey; and (3) the excretion rates of the urinary metabolites varied considerably between animals.

The pattern of urinary excretion of corticosteroids in Rhesus monkeys (Macaca mulatta).

Urinary total 17-oxogenic steroids (17-OGS), cortisol, cortisone, corticosterone, tetrahydrocortisol (THF), allo-tetrahydrocortisol (all-THF), tetrahydrocortisone (THE), cortols and cortolones, were estimated by established methods in 30 female and 20 male rhesus monkeys. The pattern of the excretion of these steroids in this species was comparable with the human corticosteroids excretion, irrespective of sex difference. The results obtained from this investigation show that they could be used during the study of adrenocortical function and cortisol metabolism in this species.

Androgen metabolism in the rhesus monkey.

A mixture of 3H-testosteron (T) and 14C-4-androstene-3, 17-dione (A) was injected intravenously into 2 (I and II) rhesus monkeys (Macaca mulatta). A third monkey (III) was injected with 3H-T only. Urine and bile samples were collected at intervals for 6 hours following the injection. The excretion, conjugation and aglycone metabolites of the steroids injected were studied using these samples. Of the injected dose, animal I (male) excreted 32% 3H and 23% 14C in the bile and 30% 3H and 21% 14C in the urine in 6 hours. Animal II (female), however, had a comparatively higher biliary excretion (66% 3H, 40% 14 C), but a urinary excretion (18% 3H, 13% 14C) comparable to that of animals I and III. The averages in the bile of the 3 animals were: unconjugated compounds 3%, glucosiduronates 78%, sulfates 9%, sulfoglucosiduronates 5% and disulfates 3%; and in urine, 5% unconjugated, 92% glucosiduronates and 3% sulfates. The aglycones obtained following hydrolysis were separated gy chromatography on Lipidex 5000, further purified by thin layer and paper chromatography and identified by co-crystallization. The major matabolites from 3H-T were androsterone and 5beta-androstane-3alpha,17beta-diol, whereas that from 14C-A was androsterone. Other metabolites identified were: etiocholanolone (3beta-hydroxy-5-beta-androstan-17-one); T, epitestosterone (epi-T), (17alpha-hydroxy-4-androsten-3-one); epiandrosterone (3-beta-hydroxy-5alpha-androstan-17-one) and 5alpha-androstane-3alpha, 17beta-diol. The results indicate that while androgen metabolism in the rhesus monkey is similar to that of the baboon and human in conjugate and metabolite formation, the rate of excretion was significantly different, resembline more closely that of the baboon than the human.

Biosynthesis of the blood-group-B-specific trisaccharide in a rhesus monkey.

A Rhesus monkey, serologically grouped as B, has been shown to excrete low-molecular-weight carbohydrate material in urine closely related to that found in human urine. Galactose feeding resulted in the excretion of a trisaccharide which was shown to be identical to the trisaccharide isolated from the urine of group B humans under the same conditions. Experiments in which [14C]galactose was administered both orally and via an intestinal vein demonstrated that the intestine is the site of biosynthesis.

Urinary metabolites of 2, 5, 2', 5'-tetrachlorobiphenyl in the nonhuman primate.

The metabolism of 2, 5, 2', 5'-tetrachlorobiphenyl (TCB) in nonhuman primates was found to be different from that previously reported in lower species. Monohydroxy TCB (I), the only metabolite in the ether extracts of rat urine, is a minor metabolite in the urine of nonhuman primates. The two major metabolites identified in the urine were dihydroxy TCB (II) and trans-3,4-dihydro-3,4-dihydroxy TCB (III). A second minor metabolite was identified as hydroxy-3,4-dihydro-3,4-dihydroxy TCB (IV). None of the above mentioned metabolites have been reported in primates and only I and III have been identified in lower animals. It is concluded that a likely mechanism for metabolism of TCB in primates is through arene oxide intermediates. This observation is of particular importance in that these types of intermediates are known to alkylate cellular components causing carcinogenic, mutagenic, necrogenic and teratogenic effects.

Amylase iso-enzymes in primates of the families Lemuridae, Cebidae, Cercopithecidae, Hylobatidae and Pongidae.

This paper presents the first data in the alpha-amylase iso-enzymes in primates of the families: Lemuridae, Cebidae, Cercopithecidae, Hylobatidae and Pongidae. By means of agar-gel electrophoresis of urine samples from 33 individuals belonging to ten species of the above mentioned families a total of 14 different variants of amylase heterogeneity were found.