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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1645
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 3 967-973
Copyright © 2008 by The Endocrine Society

The Role of the Receptor Activator of Nuclear Factor-{kappa}B Ligand/Osteoprotegerin Cytokine System in Primary Hyperparathyroidism

Inaam A. Nakchbandi, Robert Lang, Barbara Kinder and Karl L. Insogna

Section of Endocrinology (I.A.N., K.L.I.), Department of Medicine, and Department of Surgery (B.K.), Yale University School of Medicine, New Haven, Connecticut 06520; Max-Planck Institute for Biochemistry (I.A.N.) and University of Heidelberg (I.A.N.), 69120 Heidelberg, Germany; and Osteoporosis Center (R.L.), Hamden, Connecticut 06518

Address all correspondence and requests for reprints to: Inaam Nakchbandi, M.D., F.A.C.P., Max-Planck Institute for Biochemistry and University of Heidelberg, Im Neuenheimer Feld 305, 2. OG, R 210, 69120 Heidelberg, Germany. E-mail: inaam.nakchbandi{at}urz.uni-heidelberg.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The mechanisms of action of PTH on bone in vivo remain incompletely understood. The objective of this investigation was to examine changes in serum levels of receptor activator of nuclear factor-{kappa}B ligand and osteoprotegerin (OPG) in primary hyperparathyroidism and their relationship to bone loss.

Patients and Methods: Twenty-nine patients with primary hyperparathyroidism had baseline circulating soluble receptor activator of nuclear factor-{kappa}B ligand (sRANKL) and OPG measured. The relationship to biochemical markers of bone turnover and changes in bone mineral density over 2 yr was examined.

Results: Baseline sRANKL levels were elevated (1.7 ± 0.1 pmol/liter), whereas OPG remained in the normal range (5.6 ± 0.4 pmol/liter). Circulating sRANKL did not correlate with PTH but did correlate with markers of bone resorption (urine deoxypyridinoline cross-links: r = 0.51, P < 0.01; serum N-telopeptide of type I collagen: r = 0.37, P < 0.05). Furthermore, sRANKL correlated with both IL-6 and IL-6 soluble receptor (IL-6sR) (r = 0.47, P < 0.05 and r = 0.55, P < 0.005, respectively). Serum sRANKL levels also correlated with bone loss at the total femur (r = –0.53, P < 0.01). Lastly, a high value of sRANKL in combination with values of IL-6 and IL-6sR in the upper quartile (sRANKL ≥ 1.81 pg/ml, IL –6 ≥ 11.8 pg/ml, and IL-6sR ≥ 45.6 ng/ml) defined a group of four women with significantly greater rates of bone loss at the total femur than the remaining patients (–2.7 ± 1.7% vs. +0.5 ± 0.3%; n = 4 vs. n = 19, P < 0.05).

Conclusion: Determination of circulating levels of sRANKL may be useful in identifying patients with mild primary hyperparathyroidism at greater risk for bone loss. The fact that circulating sRANKL did not correlate with PTH but did correlate with markers of bone resorption suggests that skeletal responsiveness to PTH may differ in this disease.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Despite improved understanding of the mechanism by which PTH exerts its effects in bone, the mechanisms of bone loss in primary hyperparathyroidism remain incompletely understood. This is underscored by the fact that approximately 75% of patients with mild primary hyperparathyroidism lose some bone mass over time, whereas, for reasons that remain unclear, 25% do not (1, 2). The receptor activator of nuclear factor-{kappa}B ligand (RANKL)/osteoprotegerin (OPG) system is known to be critical for mediating the effects of PTH in vitro and in vivo.

In addition to the RANKL/OPG system, it has been reported that the IL-6/IL-6 soluble receptor (IL-6sR) cytokine system plays a role in mediating the effects of PTH in vitro and in vivo. Experiments in vitro have shown that PTH stimulates the production of IL-6 by bone cells (3). Both IL-6 and IL-6sR are important for osteoclast formation (4). In a prospective, observational, longitudinal study conducted over a period of 2 yr, the serum levels of IL-6sR correlated significantly with the degree of bone loss at the total femur in patients with primary hyperparathyroidism, and a combined elevation in both IL-6sR (≥45.6 ng/ml) and IL-6 (≥11.8 pg/ml) was associated with a significantly higher rate of bone loss in comparison with subjects with an elevation in only one or neither cytokine (annual bone loss rates: –2.6 ± 1.3 vs. +0.4 ± 0.3%; P < 0.05) (5).

Despite these findings, not all data support an important role for IL-6/IL-6sR in mediating the resorptive actions of PTH. Thus, PTH infusion in IL-6 knockout mice results in an increase in markers of bone resorption despite the complete absence of IL-6, suggesting that PTH effects are not all mediated by the IL-6/IL-6sR system (6). In addition, mice fed a low-calcium diet to induce secondary hyperparathyroidism did not show a change in levels of IL-6 expression in bone, whereas RANKL mRNA expression was induced (7).

Expression of RANKL is induced by PTH and stimulates the formation of active osteoclasts (8, 9). The actions of RANKL are modulated by the production of the inhibitory decoy receptor OPG (10). Experiments in vitro and in vivo have shown a key role for RANKL for the formation of osteoclasts (8, 9, 10), but the exact identity of the cells producing RANKL in bone remains uncertain (11). Evidence that serum levels of soluble RANKL (sRANKL) may reflect changes in the activity of PTH comes from the work of Buxton et al. (12). These investigators reported that the improvement in bone mineral density (BMD) seen in patients with glucocorticoid-induced osteoporosis treated with an anabolic PTH regimen was associated with changes in serum levels of sRANKL and OPG, suggesting that the levels of these two molecules may reflect the status of bone turnover in response to PTH.

The relationship between IL-6/IL-6sR and RANKL appears complex. On one hand, IL-6 can induce RANKL production in bone, suggesting that the IL-6/IL-6sR system may lie upstream of RANKL in a cytokine cascade PTH->IL-6/IL-6sR->RANKL (13, 14). On the other hand, in vitro evidence suggests that IL-6 may act independently of RANKL to induce osteoclastogenesis (4, 15). Finally, RANKL has been reported to induce osteoclast formation in the absence of IL-6 (7).

The aims of this study were to: 1) examine the effect of primary hyperparathyroidism on circulating levels of sRANKL and OPG, 2) examine the relationship between the IL-6/IL-6sR and RANKL/OPG cytokine systems in subjects with primary hyperparathyroidism, and 3) determine whether serum levels of sRANKL and/or OPG are useful predictors of bone loss in patients with primary hyperparathyroidism.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and study protocols

Twenty-nine patients with primary hyperparathyroidism who have been previously described were included in the biochemical and BMD analyses for this study (5). Three subjects underwent curative surgery after the first study visit and three more study subjects were followed up for only 6 months. These six patients were excluded from the longitudinal analyses correlating change in BMD with circulating cytokine levels.

At baseline and every 6 months thereafter, fasting blood was drawn and 2- and 24-h urine specimens collected. The 2-h urine samples were collected in the morning in the fasting state usually between 0800 and 1000 h. Bone density at the femur, lumbar spine, one third wrist site, and total body were measured every 6 months with a Hologic 4500 machine (Hologic, Bedford, MA). The Hologic 4500 uses fan beam technology. Only BMD data for patients followed up for at least 1 yr were included in the analyses. As noted, six patients were not included in the BMD analyses.

Measurements

Cytokines Serum levels of sRANKL and OPG were measured by highly sensitive solid-phase ELISAs (Alpco Diagnostics, Windham, NH). The lower limits of detection for these assays are 0.08 pmol/liter for sRANKL and 0.14 pmol/liter for OPG. The precision of these assays according to the manufacturer for sRANKL are: intraassay coefficient of variation (CV), 5%, interassay CV, 9%; and for OPG: intraassay CV, less than 10%, interassay CV, less than 10%. The normal range in our laboratory for sRANKL is less than 1 pmol/liter and for OPG, 4–6.5 pmol/liter. The control population consisted of 23 healthy controls who were comparable with the group included in the BMD analyses. Serum levels of IL-6 and IL-6sR were measured previously in these samples (5) and are reported again here solely for the purposes of comparison to the serum levels of sRANKL and OPG.

Markers of bone turnover

Serum N-telopeptide of type I collagen (NTX) was measured by ELISA (Ostex, Seattle, WA), with a sensitivity of 3.2 nM bone collagen equivalents (BCE). The intraassay CV for this assay in our laboratory is 4.2% and the interassay CV is 5.4%. Urine deoxypyridinoline cross-links (DPD) were measured by ELISA (Metra Biosystems, Mountain View, CA). The CVs for these measurements in our laboratory are an intraassay of 4.3% and an interassay of 4.6%. Urine DPD was corrected for urinary creatinine, which was measured by a colorimetric method using alkaline picrate solution. The sensitivity of the urine DPD assay was 1.1 nM/mmol creatinine. The normal ranges as provided by the respective manufacturers are: serum NTX, 6.2–19.0 nM BCE, and urine DPD, 3.0–7.4 nM/mmol creatinine.

BMD analyses

BMD at the femur, lumbar spine, 1/3 wrist site, and total body was measured every 6 months using a Hologic 4500 machine and then independently reviewed by two trained physicians. A reading was omitted only when suggested by both physicians. One patient’s femoral neck and another patient’s 1/3 wrist site BMD were omitted in the final analyses on the recommendation of both densitometrists because of obvious repositioning inconsistency. Changes in BMD were expressed as an annual percentage change from baseline using both the raw BMD values and the machine calculated averaged rates of change. The machine-calculated averaged rates of change are the data presented in the text and graphically.

Statistical analyses

All analyses were performed using the SPSS Statistical Package (version 11.0, Chicago, IL). Bivariate correlations between biochemical variables were assessed using Pearson’s r value. Linear regression analyses were used to compare both cytokine systems. Based on prior published data, the working hypothesis was that a proresorptive marker would correlate positively with rates of bone loss; as a consequence one-tailed analyses were used in this context only. Results are expressed as mean ± SEM.

Informed consent

The study was approved by the Yale University Human Investigation Committee. All subjects gave informed written consent.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Relationships among serum PTH, sRANKL, and OPG

In the 29 patients with primary hyperparathyroidism, mean serum levels of sRANKL were elevated (1.7 ± 0.1 pmol/liter; normal < 1 pmol/liter), whereas OPG remained within the normal range (5.6 ± 0.4 pmol/liter) (Table 1Go). In the nine women who were receiving hormone replacement therapy (HRT) mean sRANKL levels were lower than the mean value in the 17 estrogen-deficient women (1.5 ± 0.1 vs. 1.8 ± 0.1 pmol/liter; P < 0.05). There was no significant difference in mean OPG levels in women receiving HRT, compared with those not taking estrogen (5.5 ± 0.7 vs. 5.7 ± 0.5 pmol/liter; P = 0.82).


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  		TABLE 1. Baseline characteristics for the patients with primary hyperparathyroidism as previously reported (5 ), with modifications

 
Neither sRANKL nor OPG correlated with PTH, nephrogenous cAMP or 1,25 dihydroxyvitamin D3 [1,25(OH)2D] in the whole group. Similarly, sRANKL and OPG did not correlate with PTH, nephrogenous cAMP, or 1,25(OH)2D when the data from women receiving HRT and estrogen-deficient women were analyzed separately.

Relationship of RANKL and OPG with markers of bone turnover

In the group as a whole, sRANKL correlated significantly with both urine DPD and serum NTX (r = 0.51, P < 0.01 for urine DPD; r = 0.37, P < 0.05 for serum NTX; Fig. 1Go, A and B). However, the correlation between sRANKL and urine DPD was no longer significant when the subject marked a (Fig. 1AGo) was removed from the analysis, whereas the correlation with serum NTX remained significant. When the three men were excluded from the analysis, sRANKL no longer correlated with serum NTX. When estrogen-treated and estrogen-deficient women were separately analyzed, the associations of sRANKL with urine DPD and serum NTX were not observed.


Figure 1
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  		FIG. 1. Correlation of sRANKL with urine DPD (A) or serum NTX (B). The letter a represents the same outlier in A and B.

 
Relationship between the RANKL/OPG and the IL-6/IL-6sR cytokine systems

As reported previously, circulating levels of IL-6 and IL-6sR were elevated in the study group as a whole: IL-6, 12.1 ± 0.4 pg/ml, IL-6sR, 42.5 ± 2.1 ng/ml vs. mean values of 1.1 ± 0.1 and 25.1 ± 1.0, respectively, in eucalcemic controls (5, 16). Circulating sRANKL correlated with both serum IL-6 (r = 0.47, P < 0.05) and IL-6sR (r = 0.55, P < 0.005) (Fig. 2Go, A and B), whereas OPG was not significantly correlated with either IL-6 or IL-6sR. Similar results were obtained when the three men were excluded from the analysis.


Figure 2
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  		FIG. 2. Correlation of sRANKL with serum IL-6 in the entire study group (A), serum IL-6sR levels in the entire study group (B), or IL-6 just in the estrogen-deficient women (C).

 
As previously reported, serum IL-6 levels were similar in women receiving HRT and estrogen-deficient women (12.0 ± 0.4, n = 9 vs. 12.5 ± 0.5 pg/ml, n = 17; P = 0.50). However, IL-6sR levels were significantly lower in women taking estrogen, compared with estrogen-deficient women (37.7 ± 3.4 vs. 47.2 ± 2.5 ng/ml; P < 0.05) (5). When the two groups of women were considered separately, the correlation between sRANKL and IL-6 remained statistically significant only in the estrogen-deficient women (r = 0.49, P < 0.05) (Fig. 2CGo). When estrogen-treated and estrogen-deficient women were separately analyzed, the association between sRANKL and IL-6sR was not observed in either group.

Both IL-6sR and sRANKL correlate with changes in BMD

As reported previously, BMD declined slightly at all skeletal sites in the study group with mean yearly rates of change of –0.6 ± 0.3% at the lumbar spine, –0.2 ± 0.4% at the total femur, –0.9 ± 0.5% at the femoral neck, –0.5 ± 0.5% at the 1/3 wrist site, and –0.8 ± 0.3% at the total body (Table 2Go) (5). Serum levels of sRANKL in the group as a whole correlated with rates of bone loss at the total femur (r = –0.55, P < 0.01) but did not show significant correlations with rates of bone loss at other sites.


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  		TABLE 2. Baseline BMD data as previously published (Ref. 5 )

 
To eliminate the confounding effect of gender, we separately analyzed the data from female study subjects. In women, as in the group as a whole, serum sRANKL levels correlated with the annual rate of bone loss at the total femur (sRANKL: r = –0.53, P < 0.01) (Fig. 3Go). The correlation between sRANKL and the rate of bone loss at the total femur remained significant after excluding the subject marked by b (Fig. 3Go) (r = –0.45, P < 0.05). We previously reported that serum IL-6sR also significantly correlated with the annual decline in BMD at the total femur (r = –0.53, P < 0.01). An increase in IL-6sR by 1 ng/ml resulted in 0.1% decrease in BMD at the total femur per year, whereas an increase in sRANKL by 0.1 pmol/liter resulted in 0.4% decrease in BMD at the total femur per year. Expressed differently, an increase in IL-6sR by 1 SD was associated with a 1.1% decline in total femur BMD, and an increase in sRANKL by 1 SD was associated with a 1.9% decline in BMD at the same site.


Figure 3
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  		FIG. 3. Correlation between sRANKL and the annual change in BMD at the total femur in the entire cohort of women with primary hyperparathyroidism. The letter b represents an outlier. This is not the same outlier noted in Fig. 1Go.

 
In a linear regression model, the combination of IL-6sR and sRANKL explained a third of the variation in the yearly rate of bone loss at the total femur between the patients (adjusted R2 = 0.304, P = 0.01) (Table 3Go). In a stepwise regression analysis, sRANKL was a more robust predictor of bone loss rates than IL-6sR, explaining 27% of the variation in the yearly rate of bone loss at the total femur between the patients (adjusted R2 = 0.268, P < 0.01). IL-6sR no longer made an independent, significant contribution to the model when sRANKL was added to the regression analysis. Controlling for estrogen status did not markedly alter these associations (Table 3Go). Because weight has been shown to be increased in patients with primary hyperparathyroidism (17) and to correlate with circulating levels of IL-6 (18), we also included weight as a variable in the regression analysis. Controlling for weight did not markedly affect the association of sRANKL with bone loss (Table 3Go).


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  		TABLE 3. Multiple regression of the annual rate of bone loss at the total femur against analyzed variables in the model

 
Subjects with sRANKL values in the upper quartile (≥1.81 pg/ml) defined a subgroup in whom there was a significant decline in bone mass at the total femur, compared with the remainder of the group in whom bone density did not change (–1.7 ± 1.0 vs. +0.4 ± 0.3%; n = 7 vs. n = 16, P < 0.05). In the original publication, we reported that the upper quartile patients in whom both serum IL-6 and IL-6sR levels were highest (an IL-6sR ≥ 45.6 ng/ml and an IL-6 ≥ 11.8 pg/ml) experienced greater rates of bone loss at the total femur than the remainder of the group in whom bone mass remained relatively stable (–2.6 ± 1.3 vs. +0.4 ± 0.3%; n = 6 vs. n = 17, P < 0.005). Including only the patients with an IL-6sR 45.6 ng/ml or greater, an IL-6 11.8 pg/ml or greater, and a sRANKL 1.81 pg/ml or greater defined a group of four patients with the greatest degree of bone loss at the total femur: –2.7 ± 1.7 vs. +0.5 ± 0.3%; n = 4 vs. n = 19, P < 0.05. In this group, PTH levels were not significantly higher than the remainder of the group (112 ± 24 vs. 103 ± 10 nleq/ml, n = 4 vs. n = 17, P = NS).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The principal findings of this study are: 1) serum levels of soluble RANKL are elevated in subjects with mild primary hyperparathyroidism and correlate with markers of bone resorption, whereas levels of OPG are within the normal range and show no relationship to markers of bone resorption; 2) women with mild primary hyperparathyroidism not receiving estrogen had higher circulating levels of sRANKL than estrogen-replaced women; 3) circulating levels of sRANKL correlated positively with rates of bone loss at the total femur in the subjects with primary hyperparathyroidism; 4) sRANKL alone explained 27% of the loss in BMD at the total femur; 5) an increase in IL-6sR by 1 SD results in a yearly decrease in total femur BMD of 1.1%, whereas an increase in sRANKL by 1 SD results in a yearly decrease in femur BMD of 1.9%; and 6) the combined elevation of IL-6, IL-6sR, and sRANKL identified a group with a markedly elevated rate of bone loss at the total femur when compared with the study group as a whole, despite the lack of difference in circulating PTH levels.

PTH is known to stimulate expression of RANKL in vitro and in vivo (19, 20, 21). The two known receptors for RANKL, RANK and OPG are key regulators of osteoclastic bone resorption in vitro (10, 22) and in vivo (8, 23). PTH regulates expression of both of these molecules (24, 25, 26). It is perhaps not surprising therefore that we observed an increase in sRANKL levels in patients with primary hyperparathyroidism. The most likely source of serum RANKL in patients with hyperparathyroidism is the bone. However, the fact that RANKL and OPG are expressed in other tissues such as the liver (27) and that PTH can exert effects on other organs besides the skeleton, including the liver (28), raises the possibility that nonskeletal tissues may also contribute to the changes observed in circulating levels of sRANKL.

Others have reported that serum levels of sRANKL are affected by PTH in vivo. Thus, Buxton et al. (12) reported that an intermittent PTH regimen results in increased circulating levels of sRANKL and interestingly a suppression of serum OPG levels. In contrast, Stilgren et al. (29) found no changes in circulating levels of sRANKL in patients with primary hyperparathyroidism before and after surgery. However, this same group found that transcripts for RANKL were increased and those for OPG were lower in bone biopsies from patients with hyperparathyroidism when compared with values in biopsies obtained after surgery. Locklin et al. (30) examined the effect of continuous PTH treatment on RANKL and OPG production by cultured human bone marrow obtained from healthy volunteers. They found that continuous exposure to PTH increased RANKL transcript expression and suppressed expression of mRNA for OPG.

A relationship between estrogen and circulating levels of sRANKL and OPG is suggested by in vivo data showing an increase in mRNA of RANK, RANKL, and OPG in the bone of ovariectomized rats, compared with controls (31). Consistent with this idea, bone marrow cells from estrogen-deficient postmenopausal women express higher levels of cell surface RANKL (32). This suggests that up-regulation of RANKL on bone marrow cells plays a role in mediating the accelerated rate of bone loss observed after estrogen withdrawal. Our data are consistent with these observations insofar as higher circulating sRANKL levels were seen in estrogen-deficient women when compared with estrogen-replaced women (P < 0.05).

Because the production of both IL-6/IL-6sR and RANKL are stimulated by PTH and because RANKL expression can be stimulated by members of the IL-6 cytokine family (33), we were interested in the relationships among IL-6/IL-6sR, RANKL, OPG, and bone loss in patients with primary hyperparathyroidism. We found that RANKL correlated with rates of femoral bone loss in our study group. A relationship between the levels of circulating RANKL or OPG and bone mass has been observed in other diseases. In chronic liver disease, for example, an elevation in the ratio of RANKL/OPG was found to correlate with osteopenia or osteoporosis at the femoral neck and lumbar spine (27). Similar associations have been reported in inflammatory bowel disease (34), rheumatoid arthritis (35, 36), and spinal cord injury (37).

Our finding that RANKL is a better predictor than IL-6/IL-6sR for bone loss is consistent with the acknowledged central role of RANKL in modulating osteoclast formation because RANKL by itself is able to support osteoclast formation under certain experimental conditions, whereas IL-6 alone is not (4, 38). The interdependence of the IL-6/IL-6sR and the RANKL/OPG systems, however, makes dissecting the exact contribution of each molecule in this setting difficult (33).

Our data suggest that serum cytokine levels may be more reliable predictors for rates of skeletal loss in hyperparathyroidism than levels of PTH itself. Furthermore, our data suggest that the cytokine response to comparable levels of PTH might differ among patients, modified by factors such as estrogen status, as our data demonstrate. Nonetheless, the hypothesis that the skeletal response to PTH may vary in hyperparathyroidism despite similar circulating hormone values remains unproven and controversial.

Our study has some limitations. Our sample size is small, and the duration of follow-up relatively short (22 ± 1.5 months). In addition, the study was originally designed to examine serum IL-6 and IL-6sR as the principal outcome variable. The examination of sRANKL and OPG is therefore a post hoc analysis. These data nevertheless provide insight in the physiology of sRANKL and OPG and their relationship to the Il-6/IL-6sR system in primary hyperparathyroidism that serve as a focus of future studies.

In summary, we have shown that serum sRANKL levels are elevated in patients with mild primary hyperparathyroidism and together with circulating levels of IL-6 and IL-6sR may be a useful predictor of bone loss in women with a mild form of this disease (PTH < 4-fold increased over the upper limit of normal). A larger clinical study is warranted to confirm this finding.


   Acknowledgments
 
We thank Dr. Mary Ann Mitnick in the General Clinical Research Center Core lab for performing all of the biochemical analyses.


   Footnotes
 
This work was supported by the Yale General Clinical Research Center (National Center for Research Resources Grant RR00125), the Max-Planck Society in Germany (to I.A.N.), and National Institutes of Health Grant AG15345 (to K.L.I.).

Disclosure Statement: All authors have nothing to declare.

First Published Online December 11, 2007

Abbreviations: BCE, Bone collagen equivalent; BMD, bone mineral density; CV, coefficient of variation; DPD, deoxypyridinoline cross-links; HRT, hormone replacement therapy; IL-6sR, IL-6 soluble receptor; NTX, N-telopeptide of type-I collagen; 1,25(OH)2D, 1,25 dihydroxyvitamin D3; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-{kappa}B ligand; sRANKL, soluble RANKL.

Received August 24, 2007.

Accepted November 29, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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G. El-Hajj Fuleihan
Hyperparathyroidism: Time to Reconsider Current Clinical Decision Paradigms?
J. Clin. Endocrinol. Metab., September 1, 2008; 93(9): 3302 - 3304.
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