Measuring urinary testosterone levels of the great apes--problems with enzymatic hydrolysis using Helix pomatia juice.

Helix pomatia (Hp) juice is a common enzymatic preparation for deconjugation of urinary steroids. It has been used in many published studies on urinary testosterone (T) in chimpanzees and bonobos, although the ability of Hp juice to convert other urinary steroids into T has been reported for human urine. We developed a protocol for determination of reliable T levels in primate urine using liquid chromatography-mass spectrometry. T levels were determined in a set of human, bonobo and chimpanzee urine samples (A) by measurement of intact testosterone glucuronide (TG) and testosterone sulfate (TS), (B) after hydrolysis/solvolysis with beta-glucuronidase from Hp and (C) from Escherichia coli. When samples were hydrolyzed with Hp juice, results were not correlated with the direct assay of TG and TS, and determined T concentrations were considerably higher. By contrast, hydrolysis with E. coli beta-glucuronidase yielded a good agreement of T concentrations. We demonstrated the ability of Hp juice to convert androst-5-ene-3beta, 17beta-diol (A(5)diol) into T using commercial standards and within the urine of all three species. As A(5)diol usually is present at higher levels in urine than T, this artifact leads to erroneous results for T concentrations in primate urine. The proportion of T excreted as sulfate (TS) is often neglected as TS can only be cleaved by additional solvolysis. In all three species, we found substantial amounts of TS in the urine of some subjects and a high variance of TS proportion between and within subjects. Therefore the inclusion of solvolysis into the sample preparation seems necessary.

Comparative urinary androstanes in the great apes.

Urinary androstanes from seven species of male great apes (human, bonobo, chimpanzee, lowland gorilla, mountain gorilla, Bornean orangutan, and Sumatran orangutan) were separated by HPLC and detected by RIA using two testosterone antibodies. All animals examined showed the presence of testosterone and six additional immunoreactive peaks. Although testosterone was the dominant peak (85%) in human urine, its proportion in urine was much less in the other apes, ranging from a high of 59% in the bonobo and chimpanzee to a low of 24% in the mountain gorilla. Urinary androstanes were also directly visualized using nano-spray mass spectrometry (nanoESI-MS). Although the RIA can qualitatively produce a strong signal for testosterone in unchromatographed urine, it is quantitatively present only as a trace metabolite, as demonstrated by nanoESI-MS. The combination of the two techniques showed large differences in androstane metabolism between the seven species. A previously undescribed testosterone metabolite (tentatively identified as either delta1- or delta6-testosterone sulfate) was present in significant proportions in all of the non-human apes examined. We conclude that in the great apes, testosterone is only a trace metabolite in urine, and as a consequence, its measurement may not produce results that parallel the levels of serum testosterone. The RIA measurement of urinary testosterone in part records additional androstane metabolites, which vary even between closely related genera, making the results neither equivalent with nor comparable to different species.

Urinary estrogen excretion during pregnancy in the gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus) and the human (Homo sapiens).

Urinary estrogen components were separated, identified and quantified throughout the pregnancy of the gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) and compared to estrogen levels in normal human pregnancies. Fetal and neonatal adrenals from each species were also compared in terms of weight and relative amounts of fetal zone. The results demonstrate that gorillas and chimpanzees excrete 4- to 5-fold less estrogen during pregnancy than the human and orangutan which are similar to each other. The lower estrogen excretion appears to be related to a smaller fetal adrenal in both the gorilla and chimpanzee which reveal both a reduced adrenal weight and increased definitive to fetal zone ratio when compared to either the human or orangutan.

Pregnancy monitoring in diverse primate species by estrogen and bioactive luteinizing hormone determinations in small volumes of urine.

Immunoreactive estrogen and luteinizing hormone/chorionic gonadotropin (LH/CG) bioactivity have been measured throughout pregnancy in diverse primate species with a sample methodological approach utilizing small volumes of urine collected randomly. Total immunoreactive estrogen levels and the relative abundance of component estrogens (estrone, estradiol and estriol) at various stages of gestation were compared in the human, orangutan, pygmy chimpanzee, Douc langur and capuchin. Measurement of urinary LH/CG bioactivity enables an earlier detection of pregnancy when compared to the nonhuman primate pregnancy test. The methods described provide a practical and non-stressful way to detect and monitor pregnancy in widely divergent primate species.

Pregnancy diagnosis in the orangutan (Pongo pygmaeus) using the subhuman primate pregnancy test kit.

A determination was made of the gestational interval over which the Subhuman Primate Pregnancy Test, a hemagglutination inhibition test for urinary chorionic gonadotropin, accurately indicated conception and the continuation of pregnancy in an orangutan (Pongo pygmaseus). The initial positive diagnostic test response occurred about 8 months before parturition and positive responses continued throughout gestation. A test made one day after parturition was positive indicative of some residual urinary chorionic gonadotropin. Tests made 3 days after parturition and later were negative.

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.