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Technical Briefs |
1
Endocrinological Laboratory,
2
Department of Child and Adolescent Psychiatry, and
3
Central Diagnostic Laboratory, University Medical Center Utrecht, KC03.063.0, PO Box 85090, 3508 AB Utrecht, The Netherlands
a author for correspondence: fax 31-30-250-5378, e-mail J.Thijssen{at}Lab.AZU.NL
In psychobiological and psychopathological studies, biological markers are used as indicators for changes in neurotransmitters in the central nervous system. The concentration and/or turnover of serotonin, dopamine, glutamine, and other neurotransmitters can partly be deduced from information obtained on the concentrations of hormones in blood after specific stimulation. This "endocrine window" is of major importance for studies in the field of biological psychiatry.
More recently, it has been discovered that the measurement of certain hormones in saliva can be used as a good reflection of the plasma or serum concentrations of these hormones, which certainly seems to be true for steroid hormones such as cortisol, androstenedione, 17-hydroxyprogesterone, and testosterone. Saliva can be collected more easily and more frequently than blood, and its collection causes much less stress. Cortisol measurements in saliva in particular have received much attention.
In a very recent publication, the concentration of salivary prolactin was described as a marker of central serotonin turnover in rhesus macaques (1). In this report, saliva concentrations of prolactin between 4 and 17 µg/L were described, concentrations which are similar to those in the plasma/serum of human subjects. Although any addition to the possibilities for obtaining information on central neurotransmitters is of great practical importance, we had reservations with respect to the permeability of the human salivary cell membrane to relatively large molecules such as prolactin. Therefore, we decided to assess the concentrations of prolactin in human saliva. In a pilot study, we were unable to measure prolactin in saliva, using an assay (Abbott AxSYM) that is capable of measuring 0.2 µg/L prolactin and therefore has a detection limit that is at least 10-fold lower than the assay used by Lindell et al. (1).
Because of these results, we conducted a more systematic study on salivary prolactin concentrations in humans. For that purpose, we used four widely used diagnostic techniques to determine prolactin in salivary samples from pregnant women and prepubertal boys, who have a very high and a very low serum prolactin, respectively. To avoid so-called matrix effects (2), serum/saliva mixtures were included in the analyses.
Individual saliva samples from 10 pregnant women, collected during the third trimester of pregnancy, were used. These specimen had been collected as part of a systematic study on cortisol concentrations during the course of an uncomplicated pregnancy. The samples had been collected in the morning, put in ice within minutes, frozen within 1 h of collection, and stored at -20 °C until the women had delivered a healthy child. Randomly chosen samples from 10 individuals were used. A second collection of samples had been obtained during studies on conduct disorder prepubertal boys and normal controls, follow-up studies of those reported earlier (3)(4). These samples had been collected similarly and stored at -20 °C; samples of 10 controls were chosen at random. These samples had been collected in studies approved by the hospital’s ethics committee. All samples had been frozen and thawed one time before our experiments, and the samples had been stored for <2 years after collection. From each of these 20 individual saliva samples, a 1:1 (by volume) mixed sample was prepared with a low-prolactin serum pool.
Serum samples with previously measured prolactin concentrations were obtained from the diagnostic laboratory. A mixed serum pool was prepared from four individual samples with low (<5 µg/L) prolactin concentrations. Results are expressed in µg/L; conversion factors from IU/L to µg/L were used as stated by the manufacturers (see below).
Prolactin was measured using four different sets of reagents on four discrete analyzers that were available in the hospital. All final determinations were carried out in one series; reagents and analyzers were used in accordance with the instructions of the manufacturers. All four sets of prolactin reagents were calibrated against the 3rd International Standard for prolactin, WHO 84/500. Conversion factors from IU/L to µg/L were 24 for the Abbott assay, 21.5 for the Bayer assay, and 21.2 for both the Diagnostic Products Corporation (DPC) and Roche assays.
The standard technique for the determination of prolactin in our hospital uses the Abbott AxSYM (Abbott Laboratories); three other widely used routine methods have also been tested: the Advia Centaur (Bayer Diagnostics Division), ES300 (Roche Diagnostics) and Immulite (DPC).
The stability of prolactin in saliva was tested by adding 10 µL of prolactin standard to 500 µL of freshly collected saliva and measuring its recovery after freezing/thawing and after storage during 60 h at -20, 4, and 20 °C. The loss of prolactin after freezing/thawing was 2% ± 1% (n = 5). Storage at -20 °C caused no change in prolactin; losses of 10% ± 2% and 30% ± 2% were observed after storage at 4 and 20 °C, respectively.
We compared the four techniques, using six serum samples with prolactin
concentrations of 3–40 µg/L; the results obtained with each of the
methods in these sera (serum pool and human sera A–E) are shown in
Table 1
. All methods showed good agreement: the correlation
coefficients were 0.995–0.997. Absolute concentrations did show some
differences for prolactin concentrations between the methods: the
slopes of the calculated regression lines were 0.91–1.09, with
intercepts of -0.5 to 1.2 µg/L.
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Our measurements in saliva consistently showed very low to undetectable
prolactin concentrations. The results are presented in Table 1
, which
shows the mean prolactin values for each of the four groups of samples
that were evaluated. For each of the four methods used, one to three of
the pure saliva samples per group did show a prolactin concentration
above the lower limit of detection for that method;
however, no difference was found in the frequency of detectable
concentrations between samples from pregnant women or prepubertal boys.
The detection limits of the methods were not identical, the
most sensitive was the AxSYM (<0.2 µg/L), whereas the Bayer and DPC
methods were unable to detect concentrations <0.5 µg/L. The limit of
detection had been tested by running 10 replicates of the zero
calibrator of each set of reagents in the same series of assays as the
samples. The detection limit was estimated at 2 SD of this calibrator;
the results are given in Table 1
.
Because all of the methods studied were developed for the analysis of
prolactin in or serum samples, a possible bias had to be excluded. For
that purpose, a 1:1 mixture of saliva and the low serum pool was
prepared and measured; the results are shown in Table 1
. In the Abbott,
Bayer, and DPC methods, very good agreement could be seen between the
prolactin concentration in the undiluted low-prolactin serum pool and
the concentration after dilution with saliva. In these cases, the
values of the mixed samples were very close to the expected 50% value
of the serum pool, supporting the finding that no detectable prolactin
concentrations could be found in the pure saliva samples. The Roche
method did not show a correct dilution profile: values in the mixed
samples were lower than expected, indicating some bias for that
particular method at this very low concentration. In addition, the
results with this method do not indicate detectable prolactin
concentrations in human saliva.
The recently reported observation of a biologically significant concentration of prolactin in saliva in rhesus macaques (1) could thus not be extended to humans. On the contrary, our results indicate that with these four widely used reagents for the specific and sensitive determination of prolactin in serum, no prolactin could be detected in human saliva.
According to the published information about the method used in the study by Lindell et al. (1), which gives 3.8 µg/L as the lowest detectable prolactin concentration, the detection limits of the four methods used in our study are substantially better, i.e., between 0.2 and 0.5 µg/L. It is conceivable that the methods used in our study are much more specific for the assessment of prolactin because all of them use very specific, double-antibody-based sandwich assays compared with the competitive single-antibody-based assay used in the study by Lindell et al. (1).
On the basis of our results, it seems justified to conclude that prolactin concentrations in human saliva are probably <0.2 µg/L. The results as reported by Lindell et al. (1) can of course be explained simply by differences between the two species involved. A second interesting possibility that cannot be excluded is the existence of cross-reacting peptides in the saliva of rhesus macaques. There are indications that prolactin is capable of inducing the synthesis of specific peptides in the salivary glands of rats (5), thereby inducing substances that possibly may be related to prolactin in the body. In the previous study (1), a relationship was observed between the measured substance(s) and the central serotonin turnover; this topic therefore deserves further investigation because the supposed cross-contaminants may be of great importance for future psychobiological research.
We conclude that with the four commercially available methods in our study, no prolactin is detectable in human saliva.
References
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