Serum tryptophan, kynurenine, phenylalanine, tyrosine and neopterin concentrations in 100 healthy blood donors

Introduction

Immune activation and inflammatory responses play a relevant role in the pathogenesis of a variety of diseases like cancer, infections and autoimmune disorders. Concentrations of 6-(D-erythro-1′,2′,3′-trihydroxypropyl)-pterin (neopterin), a well-established immune activation marker, were found to be elevated in these patients and to be of diagnostic value [1, 2]. Neopterin is primarily produced and released from activated human monocyte-derived macrophages, dendritic cells and astrocytes [3–5]. Thereby, formation of increased amounts of neopterin in macrophages is mainly initiated by Th1-type cytokine interferon-gamma (IFN-γ) [3], whereas type I interferons are also potent inducers of neopterin production in other cells like dendritic cells or astrocytes [4, 5]. The central enzyme involved in the biosynthesis of neopterin is GTP-cyclohydrolase-1 (GCH-1), which converts guanosine triphosphate (GTP) to 7,8-dihydroneopterintriphosphate [6].

IFN-γ is also the most important inducer of the antiproliferative and immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) [7, 8]. IDO converts the essential amino acid L-tryptophan (TRP) to kynurenine (KYN), which is the first and rate-limiting step of tryptophan catabolism. IDO enzyme activity can be calculated by using the KYN to TRP quotient. Accelerated TRP breakdown and elevated KYN/TRP concentrations have been shown in many diseases and closely correlate with concentrations of immune activation markers like neopterin [1, 9–12]. In these situations, inflammation-activated IDO is driving the TRP catabolism and not tryptophan dioxygenase, a hepatic enzyme that is regulated via tryptophan levels or glucocorticoids. In many cases, the TRP breakdown rate not only correlates with neopterin concentrations, but also with the extent and the activity of the disease, e.g., in viral infections or malignant tumors [8–13].

While in human monocytic cells enzyme GCH-1 is responsible for the production of neopterin, it also induces the synthesis of 5,6,7,8-tetrahydrobiopterin (BH₄), an essential cofactor of aromatic amino acid monooxygenases such as phenylalanine 4-hydroxylase (PAH), tyrosine 3-monooxygenase, tryptophan 5-hydroxylase, and also of the nitric oxide synthases (NOS) and of alkylglycerol monooxygenase [14–16]. PAH converts the essential amino acid phenylalanine (PHE) to tyrosine (TYR), which is further hydroxylated by tyrosine 3-monooxygenase to L-3,4-dihydroxyphenylalanine, a precursor of the neurotransmitter dopamine. Tryptophan 5-hydroxylase converts TRP to 5-hydroxytryptophan, the precursor molecule of the neurotransmitter serotonin. NOS produces nitric oxide (NO) by converting L-arginine to L-citrulline.

Decreased phytohaemagglutinin activity leads to an increase of PHE concentrations and of the phenylalanine to tyrosine ratio (PHE/TYR). PHE/TYR is well known to be highly elevated in patients suffering from inborn errors of PHE metabolism, e.g., classical phenylketonuria. In addition, a moderate form of hyperphenylalaninaemia is reported for patients who suffer from chronic inflammatory diseases. Notably, in such patients the increased PHE and PHE/TYR concentrations were also found to correlate with neopterin levels [17–19].

The aim of this investigation was to establish reference values for the above mentioned interrelated biomarkers in a reasonably sized healthy population of 100 blood donors of both genders, who had passed anamnestic examination and therefore can be considered as widely healthy and free from overt infections. In addition, potential associations between the concentrations of the different biomarkers were examined.

Materials and methods

Results

One hundred subjects were included in the study. The mean age of participants was 49±11.4 (mean±SD) years; 52 persons were older than 50 years (18 older than 60 years). Further demographic information is given in Table 1, as are the determined concentrations of neopterin, nitrite and amino acids. Seven blood donors presented with neopterin concentrations above the upper limit of the normal for age of 19–65 years (95th percentile), which is 8.7 nmol/L [23]. Two donation samples contained neopterin concentrations higher than 10 nmol/L; the threshold has been defined earlier as the 98th percentile in blood donors [2]. More information on the distribution of concentrations of the other parameters tested is given in Table 1.

Forty-two participants were women with a mean body mass index (BMI) of 25.4±4.91 kg/m²; the 58 males had a mean BMI of 25.9±3.30 kg/m². In Table 2, more group comparisons regarding gender are shown. Men were older, taller and heavier than females (all p<0.001). TRP and TYR concentrations were higher in males than in females (p<0.02). Also, PHE concentrations were slightly elevated in males compared to females, but the difference failed to reach the level of significance (U=1.777. p=0.075).

For the whole set of participants, a significant correlation existed between the BMI and TRP concentrations (rs=0.237; p<0.02). However, when analyzing male and female data separately, only in females but not in males, a significant correlation between body mass and the concentrations of amino acids TRP (rs=0.332), TYR (rs=0.318) and PHE (rs=0.306; all p values <0.05) was apparent (Figure 1).

Figure 1:

Scattergrams of body mass vs. phenylalanine concentrations in 42 female (upper) and 58 male (lower) blood donors (females rs=0.306, p<0.05; males rs=–0.061. p=not significant).

No significant correlation was found for BMI and neopterin concentrations and also not for age and neopterin (rs=0.012), KYN/TRP (rs=–0.028) or PHE/TYR (rs=–0.153; all not significant) concentrations in the whole data set including male and female data.

Neopterin concentrations and tryptophan metabolism were significantly associated in the whole data set (Table 3); neopterin correlated with kynurenine (rs=0.368) and KYN/TRP (rs=0.453; both p<0.001). Also, PHE/TYR (rs=0.236; p=0.018) correlated with neopterin concentrations, and there was an inverse correlation between tyrosine and neopterin concentrations (rs=–0.267; p<0.001). In both females and males, a significant correlation existed between neopterin and KYN/TRP, and interestingly in females the correlation (rs=0.621, p<0.001) was substantially stronger than in males (rs=0.366, p=0.005).

Discussion

The average neopterin concentrations observed in the 100 blood donations compare rather well with yet published data. Neopterin concentrations in males and females were not different, and also the 95th and 98th percentiles agree rather well with earlier reported values [23, 24], where similar immunoassays (radio-immunometric assays or ELISA systems) were used. However, concentrations estimated via HPLC or liquid chromatography-mass spectrometry analytics tend to be higher [25]. Most probably, this discrepancy depends on the precipitation method applied for serum protein separation. When acidic milieu is used, partial oxidation may occur, which contributes to higher neopterin levels due to oxidation of 7,8-dihydroneopterin to neopterin [26]. Only separation of unwanted serum protein with molecular sieves or precipitation with acetonitrile (CH₃CN) could be employed as alternative techniques that are not associated with 7,8-dihydroneopterin oxidation. Also, minor differences may depend on the distinct analytical procedures, e.g., the manual workup as was done in this study in contrast to the automated pipetting systems used in larger studies [24].

Concentrations of amino acids are in the expected range, although the comparison with data from the literature can be more difficult due to the fact that methods used for measurements differ widely. In most recent analyses, HPLC or other liquid chromatography methods are applied. However, common protocols include detection of analytes after derivatization reaction with ninhydrin [21], whereas in this study the natural fluorescence of compounds (for PHE and TYR measurements: excitation wavelength 210 nm and emission wavelength 302 nm; TRP: excitation wavelength 286 nm and emission wavelength 366 nm) was used, and only the kynurenine was detected via UV absorption at 360 nm.

Differences between concentrations in male and female donors are apparent: Lower TRP, PHE and TYR concentrations were observed in females as compared with males. Thereby, the TRP results agree well with earlier observations [20]. In the whole data set there existed correlations between body mass and levels of several amino acid, but gender appears to be an additional factor influencing amino acid concentrations, because there exists a correlation between body mass and amino acid levels in females but not in males. Previous investigations showed a correlation between age and neopterin and KYN/TRP [27, 28], which was not observed in this study. In this regard, the rather narrow age range of donors in this current study might be of relevance; the maximum age of our study participants was 70 years. Further, slight differences in the correlation of the immune biomarkers neopterin and KYN/TRP were found in female and male data.

Because it is well documented that immune activation influences the metabolism of the amino acid TRP and also of PHE and TYR, results of this study support the hypothesis that low-grade inflammation is responsible for the significant correlations found between immune system biomarkers like neopterin, KYN/TRP or PHE/TYR. Increased KYN levels and an elevated KYN/TRP are closely linked to accelerated TRP breakdown due to activation of the enzyme IDO [10, 11]. Deprivation of TRP is an efficient strategy during the Th1-type immune response to counteract unwanted proliferation of pathogens, infected cells and tumor cells [29]. The disturbed PHE metabolism and thus higher PHE/TYR, also correlating with immune activation, probably results from disturbed activity of PHE-metabolizing enzyme PAH, due to insufficient supply with its cofactor BH₄, which is sensitive to oxidation [17].

The alterations of amino acids concentrations found in the present study indicate that these metabolic disturbances could be of relevance not only in patients who suffer from diseases with overt immune activation and inflammation but also in healthy individuals. At least, in elderly people it was already observed that the disturbed TRP, PHE and TYR metabolism was associated with signs of depression and/or neurovegetative symptoms [30].

Nitrite concentrations showed large inter-individual variability and did not correlate with neopterin concentrations. Aside from many other factors, nitrite concentrations can be influenced by endogenous NOS activity, which oxidizes L-arginine to L-citrulline thereby releasing NO. However, in contrast to rodents, human macrophages are not a major source for inducible nitric oxide synthase derived NO [31]. An important exogenous factor influencing serum nitrite concentrations is food. Diet containing high-nitrate vegetables may heavily exceed recommended daily intake values [32]. Commensal bacteria in the oral cavity are able to reduce nitrate to nitrite [33], and also, mammalian xanthine oxidoreductase was shown to be able to reduce nitrate [34]. A number of mammalian enzymes and redox active compounds can convert nitrite into NO, and vice versa. While these new findings further put into question to assess serum nitrite in regard to immune activation, recent literature discusses the potential of a high-nitrate diet as an alternative nitrate/NO source to lower blood pressure [32, 35].

In conclusion, the establishment of the normal range measured in a reasonably high number of healthy controls provides the basis for further studies of the highly relevant topics around the metabolism of aromatic amino acids. Most importantly, the associations found in healthy donors may indicate that there exists a strong interference between immune status and amino acid metabolism that is of relevance not only in patients but also in the healthy population. This is of special interest in the chronic situation, as also prevailing minor changes in amino acids may lead to clinical relevant symptoms. In particular, this may be of relevance for the later development of especially neuropsychiatric symptoms associated with immune response [36, 37].