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Enhanced serum concentrations of parathyroid hormone (PTH) increase bone resorption. This results in elevated levels of serum calcium (Ca) and decreased bone density . Furthermore, increased levels of inflammation may be observed in patients with primary hyperparathyroidism (PHPT). Elevated concentrations of inflammatory markers such as high-sensitivity C-reactive protein (CRP) and tumor necrosis factor-α were demonstrated in some  but not all studies . In the majority of cases, PHPT is caused by either single adenomas or glandular hyperplasia producing excessive amounts of PTH. Parathyroidectomy has a cure rate as high as 90–95% . Postoperatively there is a reversal of bone loss , but follow-up of patients 2 years after surgery has not shown beneficial effects on known inflammatory markers [2, 6].
It was shown in a long-term follow-up study that PHPT patients are at increased risk of death from cardiovascular disease (CVD) . Inflammatory pathways are centrally involved in atherogenesis and atherosclerotic plaques are infiltrated by activated macrophages and lymphocytes, which probably contribute actively to disease progression . In patients with stable angina pectoris, the monocyte-activating cytokine interferon-γ (IFN-γ) has been linked to acute atherosclerotic complications including major coronary events and death [9, 10]. As IFN-γ has a short half-life, other circulating markers are often used as indicators of the activity of this cytokine. The conversion of tryptophan to kynurenine is mediated through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). Enhanced levels of IFN-γ lead to increased IDO activity and thus an increase in the kynurenine-to-tryptophan ratio (KTR) . As IFN-γ enhances the production of neopterin by activated macrophages, neopterin is another marker of IFN-γ activity .
The kynurenine pathway of tryptophan catabolism is dependent on enzymes requiring either vitamin B6 or vitamin B2 as co-factors (Fig. 1). Pyridoxal 5′phosphate (PLP) is the most commonly used serum marker of vitamin B6 status. It is the active form of vitamin B6 and reflects the total body stores of this vitamin . Low levels of PLP have been linked to conditions associated with inflammation, such as rheumatoid arthritis, CVD and the metabolic syndrome [14-16].
Figure 1. The kynurenine pathway of tryptophan metabolism. IDO, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-dioxygenase; KTA, kynurenine transaminase; KMO, kynurenine 3-monooxygenase; Kase, kynureninase; PLP, pyridoxal 5′-phosphate; FAD, flavine adenine dinucleotide; B2, vitamin B2; B6, vitamin B6.
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We have recently reported that genes involved in inflammation were up-regulated in adipose tissue from patients with PHPT. Many of the up-regulated genes were related to infiltration and activation of macrophages, possibly through enhanced IFN-γ activity . To our knowledge, systemic markers of IFN-γ-induced inflammation have not been studied in patients with PHPT. The primary objective of this study was to investigate the IFN-γ markers KTR and neopterin as well as metabolites of the kynurenine pathway of tryptophan together with serum levels of B6 vitamers in patients with PHPT. A further aim was to determine the extent to which the levels of these biomarkers change following parathyroidectomy.
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Kynurenine levels and the KTR were higher and levels of tryptophan, anthranilic acid, xanthurenic acid and B6 vitamers were lower in the 57 patients with PHPT, compared to the levels in 20 healthy control subjects. During 6 months of follow-up in 43 PHPT patients after parathyroidectomy, concentrations of PLP and anthranilic acid increased. During the same time period, the concentration of neopterin decreased whereas the KTR was essentially unchanged. These observations suggest that IFN-γ-mediated cellular immune activation was enhanced and vitamin B6 status was altered in patients with PHPT. Within months after surgery, the vitamin B6 status appeared to be normalized and neopterin concentration reduced, but some changes in tryptophan metabolism persisted.
The observed changes in tryptophan and its metabolites, in particular the KTR, in patients with PHPT, may be explained by increased conversion of tryptophan to kynurenine catalysed by IDO or TDO . The increased KTR probably mainly reflects IFN-γ stimulation of IDO linked to increased cellular Th1 immune activation. This is supported by the strong correlation that we observed between the KTR and neopterin, which is in agreement with previous results  and explained by IFN-γ being the main inducer of both neopterin and kynurenine formation . A decrease in neopterin concentration during the 6 months after surgery may be explained by a decrease in IFN-γ activity after parathyroidectomy. Of note, levels of CRP actually increased 6 months after surgery among those patients with available samples, although levels were identical in patients and controls at inclusion. This observation is in line with earlier reports describing elevated levels of CRP and interleukin-6 (IL-6) 1 year after parathyroidectomy [3, 29]. It is possible that incomplete inflammatory recovery at 6 months after surgery may also explain the modest, nonsignificant reduction in the KTR seen in our study after parathyroidectomy.
PTH stimulates the release of inflammatory cytokines such as IL-6 from bone cells and haematopoietic cells [30, 31]. IL-6 is the primary inducer of the acute phase response of the innate immune system  and may contribute to the mild inflammation observed in PHPT patients.
The most important observation in this study is a marked reduction in circulating PLP and PL in PHPT patients; PLP was normalized 1 month after parathyroidectomy. Other B vitamins were not affected. PHPT is thus another example of the many chronic inflammatory conditions characterized by a low concentration of vitamin B6 .
The mechanisms by which PLP is decreased during inflammation are not fully understood. Chronic inflammatory diseases are complex and the causes of reduced levels of PLP may be different from those of elevated levels of CRP. In this study, we did not observe an inverse correlation between CRP and PLP, as described by others . However, our results are in line with those of a study of patients with coronary artery disease, in which lower levels of PLP were found to be associated with an increased risk of coronary artery disease, independent of levels of CPR . Supplementation with pyridoxine does not normalize levels of inflammatory markers  and does not attenuate the inverse relation between such markers and PLP . These observations from intervention studies indicate that inflammation and immune activation do not cause vitamin B6 deficiency but rather change the distribution of B6 vitamers between and within intracellular and extracellular compartments. Low vitamin B6 levels during inflammation and immune activation may reflect increased demand and cellular uptake of PLP due to quantitative changes in protein turnover and involvement of PLP in cytokine production and lymphocyte proliferation [33, 35]. Altered distribution of vitamin B6 is in line with low levels of the kynurenine metabolites anthranilic acid and xanthurenic acid in PHPT patient, as both metabolites are formed by enzymes requiring PLP as a cofactor . After surgery, we observed an increase in the level of anthranilic acid, a metabolite that has previously been shown to increase in response to pyridoxine supplementation .
In line with earlier studies , we observed higher levels of ALP in patients with PHPT. Higher serum concentrations of ALP probably reflect increased bone turnover and may be related to disease severity . Also, inflammatory processes and an increased incidence of aortic calcification have been correlated with enhanced serum levels of ALP [40-42], but whether ALP per se has a role in the inflammatory response or is a product of the inflammatory processes is not clear . It is well documented that in conditions that are accompanied by increased plasma ALP, such as bone and liver diseases, PLP levels are reduced . This is explained by hydrolysis of PLP to PL catalysed by ALP. PL is the transport form of vitamin B6 for most tissues , and enhanced ALP may therefore promote cellular uptake and affect the distribution of this vitamin. Thus, increased ALP may reduce plasma PLP, but does not explain the low level of PL that we observed in PHPT patients.
A strength of this study is the measurement of two vitamin B6 species to assess its status; this is a particular advantage under conditions of elevated ALP which may affect PLP level . Another strength is the simultaneous measurement of several kynurenines, some of which are metabolized by vitamin B6-dependent enzymes. Furthermore, the metabolic abnormalities observed in PHPT patients were monitored at three time-points during a follow-up period of 6 months after parathyroidectomy. However, only 43 of 57 (75%) patients completed the longitudinal study; the remaining patients were excluded because of either lack of postoperative blood samples or consistently elevated serum levels of corrected Ca after surgery.
Because food intake may influence plasma levels of vitamin B6  and tryptophan , a possible limitation of this study is that patients were nonfasting and we did not have information on time since the last meal. However, measurement of the ratio between kynurenines and tryptophan (KTR) may correct for possible postprandial changes . Gender distribution was uneven between the patient and control groups. This was due to strict inclusion criteria in the control group and a limited number of available blood donors. Changes in kynurenines, inflammatory markers and B vitamins were adjusted for gender to correct for this. Additionally, the results were unchanged in a subgroup analysis of women only (data not shown).
In summary, patients with PHPT had decreased vitamin B6 levels and an elevated KTR. The latter points to an increased IFN-γ-mediated cellular immune activation in these patients. Some but not all abnormalities in levels of kynurenines, inflammatory markers and B vitamins were corrected 6 months after parathyroidectomy. Low plasma vitamin B6 concentration and an elevated KTR are associated with increased risk of CVD and should be evaluated as CVD risk factors in PHPT.