INTRODUCTION

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Water and sodium metabolism is dependent upon a complex interplay of hormonal, neural, and physical mechanisms controlling renal sodium and water reabsorption (1, 2). Both ANP and the renin-angio-aldosterone system play important roles in renal sodium handling and AVP controls renal free water clearance. Water and sodium homeostasis is commonly disrupted in patients with lung cancer. Hyponatremia occurs at presentation in approximately 15% of patients with small cell lung cancer and 1% of patients with non-small cell lung cancer (3).

Ectopic production of AVP by small cell lung cancer cells plays a causal role in the development of hyponatremia in patients with this cancer (3). The role of the other hormones involved in sodium and water homeostasis has not been extensively studied in patients with lung cancer. The concept that other hormones have a role in hyponatremia is supported by the observation that patients with small cell lung cancer and hyponatremia have been identified who have no detectable AVP in their plasma or AVP mRNA or immunoreactive peptide in their tumors or tumor cell lines (4). These patients with no AVP mRNA or immunoreactive peptide in their tumors or tumor cell lines may still have AVP-mediated hyponatremia caused by baroreceptor mediated release of AVP (1, 2, 7). These lesions are likely caused by lesions in afferent nerves from peripheral baroreceptors inappropriately stimulating the release of AVP from the hypothalamus.

Evidence suggests that ectopic production of ANP mRNA may also be involved in hyponatremia in patients with lung cancer. Ectopic production of ANP mRNA in tumors and tumor cell lines from patients with small cell lung cancer and hyponatremia has been documented by Northern blot, S₁-nuclease analysis, and RNAase protection assay (4, 8). ANP has been detected in small cell lung cancer tumors and tumor cell lines by radioimmunoassay (4, 6, 9). Characterization of the peptide from small cell lung cancer tumors and tumor cell lines by gel chromatography and high performance liquid chromatography has shown the peptide to be similar to the bioactive 28 amino acid form present in the plasma (9). Plasma ANP levels have been found to be elevated in patients with lung carcinoma and hyponatremia with elevated plasma AVP levels and in patients with normal plasma AVP levels (5, 6, 9, 12, 13). Despite the extensive evidence for ectopic production of ANP, the potential physiologic role of ANP in patients with hyponatremia has not been well defined.

Atrial natriuretic peptide increases the renal excretion of sodium mediated by the ANP receptors in the kidney (1, 2, 14, 15). The hormone also decreases production of renin, angiotensin II and aldosterone (14, 15). Thus, ectopic production of ANP could contribute to hyponatremia by causing a natriuresis, negative sodium balance, and nonosmotic release of AVP because of decreased intravascular volume. In this setting, we would expect to find elevated plasma levels of renin, angiotensin II, and aldosterone in patients with ectopic production of ANP because of a relative deficit of sodium. Another potential mechanism for action of ectopically produced ANP is suppression of renin, angiotensin II, and aldosterone in the plasma leading to a negative sodium balance and hyponatremia. In this setting, we would expect to find relatively low levels of renin, angiotensin II, and aldosterone in the plasma with elevated ANP.

We sought to further characterize the role these hormones played in the development of hyponatremia of malignancy by characterizing the ectopic production of AVP and ANP and the physiologic response of these hormones to a rapid saline infusion. We studied the ectopic production of AVP and ANP by immunohistochemical studies on available tissue specimens from the patients participating in this study. In addition, tumor cell lines established from patients participating in this study were examined for evidence of AVP and ANP mRNA expression immunoreactivity. The physiologic response to an infusion of 500 ml of saline over 45 minutes was performed because it had previously been shown to increase ANP plasma levels (16). The 500 ml infusion of saline was also used to generate information about the AVP and renin-angiotensin II, aldosterone system. If the plasma levels of renin, angiotensin II, and/or aldosterone levels were elevated in patients because of ANP mediated natriuresis leading to a negative sodium balance, decreased intravascular volume, causing nonosmotic release of AVP, we expected their levels to fall with a saline infusion. If the levels of renin, angiotensin II, and aldosterone were low and stayed low with an infusion, this would suggest that the elevated ANP plasma levels were suppressing the production of these three hormones. The measurement of the sodium values in the urine with the saline infusion allowed assessment of the ability of the patients to excrete the sodium load from the saline infusion.

	METHODS

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Study Population

Patients with histologically proven previously untreated small cell lung cancer and non-small cell lung cancer were evaluated for entry into the study. The patients' pretreatment evaluation has been previously described (17) and patients were assigned an ECOG performance status and stage. Patients with small cell lung cancer were designated as having limited or extensive stage disease (17, 18) and patients with non-small cell lung cancer were designated as having stage I-IV (20). The eligibility criteria include an age older than 18 yr and no prior chemotherapy or radiation therapy. Patients with brain metastases not requiring urgent therapy were eligible for inclusion in the study. Patients whose cancer was felt to be acutely life-threatening, those with hypertension, significant myocardial disease, renal insufficiency, and symptomatic hyponatremia that did not respond to simple fluid restriction were ineligible. In addition, patients taking any medication that was known to alter sodium homeostasis, such as diuretics, antihypertensive agents, medications for congestive heart failure, demeclocycline, and corticosteroids were excluded. Patients with other active cancers, excluding superficial skin carcinomas and in situ cervical carcinoma, were not eligible.

The rationale and possible side effect of the study were explained to each patient. It was made clear that the study would be of no direct benefit to the patient. The patients were then given the opportunity to sign an informed consent for participating in this trial approved by our Institutional Review Board.

Study Design

The patient received nothing by mouth after midnight and arrived in the outpatient clinic at 8:00 A.M. A physical examination including orthostatic blood pressure and pulse rate was performed and the initial urine sample collected. The patient was then seated in upright position for the remainder of the study with intravenous access obtained in both antecubital fossae. One hour after insertion of the intravenous lines, initial blood samples would be drawn and an infusion of 500 ml 0.9% saline took place over 45 min. Vital sign measurements were performed and blood drawn every 15 min after the initial samples for 90 min. Seven samples for plasma AVP, ANP, renin activity, angiotensin II, and aldosterone were collected. Serum chemistry values (sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine) and plasma osmolality were measured from blood drawn at 0, 45, and 90 min. Urine chemistry (sodium, potassium, chloride) and osmolality were measured at 0 and at 90 min. The intravenous lines were then removed. Patients who were not willing to undergo the saline infusion with multiple plasma and urine sampling were given the opportunity to provide plasma samples for a single time for AVP and ANP determination after an overnight fast. Pathology specimens from diagnostic and staging procedures were obtained for immunohistochemical studies, molecular studies and establishment of cell lines.

Definitions

Hyponatremia was defined as three serum sodium levels of less than or equal to 130 mEq/l recorded prior to administration of chemotherapy (4, 6). Plasma hormone level upper limits of normal were determined from published literature that used normal controls. Levels of AVP were determined to be evaluated when analyzed with serum osmolality using a graph designed by Robertson and coworkers (7). Plasma ANP levels of 10 pmol/l or less were considered normal (16, 21, 22). PRA of 2.0 ng/ml/h or less were considered normal (23, 24). Plasma angiotensin II levels of 20 pmol/l or less were considered normal (22, 24). Plasma aldosterone levels of 240 pmol/l or less were considered normal (21, 22, 24, 25).

Sample Processing

Serum and urine chemistries plus plasma and urinary osmolality samples were processed by the clinical chemistry laboratory in the National Naval Medical Center, Bethesda, MD. Plasma hormone samples were collected in prepared tubes and immediately stored in ice. Blood samples for plasma ANP estimation were collected in chilled tubes with final concentrations of 4 mM EDTA, 5 micromolar pepstatin A, and 1 micromolar phenylmethyl-sulfonyl fluoride (PMSF; Sigma Chemical Corporation, St. Louis, MO). Blood samples for plasma AVP, renin activity, and angiotensin II estimation were collected in chilled tubes containing 4 mM EDTA. Samples for plasma aldosterone estimation were collected in chilled tubes with finals concentrations of 1.5 IU heparin (Lymphomed, Deerfield, IL). Tubes were centrifuged at 1,000 g at 4° C for 15 min, plasma was then removed and stored in 3 aliquots at 70° C.

Tumor Cell Line Studies

Attempts were made to establish tumor cell lines from patients with lung cancer treated on institutional review board approved protocols at the Medicine Branch (17). Tumor cell lines were also studied for AVP and ANP mRNA expression using reverse transcriptase-polymerase chain reactions. cDNA strands were generated from the mRNA as previously described (26). The primers for AVP were sense 5'-GGCCTACTGGCCTTCTCCTCC-3' and antisense 5'-CTCTCGTCGTTGCAGCAAAC which amplified a 293-bp product (27). The primers for ANP were sense 5'-CAACGCAGACCTGATGGATT-3' and antisense 5'-TTAGGAGGGCAGATCGATCAGA-3', which amplified a 236-bp product (28). Both primers amplified products that spanned the first intron of their respective genes. The polymerase chain reactions were performed using techniques previously described. AVP amplification was performed using a 4-min denaturation step at 95° C, followed by 40 cycles of 15 s at 96°, 30 s at the annealing temperature of 50° C, and 30 s at 72°. ANP amplification was performed using a 4-min denaturation step at 95° C, followed by 40 cycles of 15 s at 94°, 30 s at the annealing temperature of 55° C, and 30 s at 72°. The PCR products were resolved on agarose gels and photographed.

Approximately 0.5-1.0 ml of packed tumor cells were harvested during log phase growth and washed twice in phosphate buffered saline at 4° C. The lung cancer cell lines were then processed for measurement of immunoreactive AVP and ANP as previously described (4).

Radioimmunoassay

Plasma ANP and AVP levels were determined after the plasma was extracted using techniques previously described (4). The limit of detection of ANP was 2-5 pmol/l and AVP was 0.01 pmol/l. The interassay and intraassay variabilities have been previously reported (4). PRA was measured by radioimmunoassay according to the method described by Menard and Catt, (29) using commercial angiotensin I (Bachem, Torrence, CA), (3-[¹²⁵I]iodotyrosyl⁴) angiotensin I (Amersham International, Buckinghamshire, UK), and rabbit angiotensin I (human) antiserum (Peninsula, Belmont, CA). The limit of detection of angiotensin I was 4 pmol/l. The interassay variability for plasma renin activity was 10% and the intraassay variability was 10%. Plasma angiotensin II immunoreactivity was performed in a similar fashion to PRA radioimmunoassay using commercial angiotensin II (Bachem, Torrence, CA), (3-[¹²⁵I]iodotyrosyl⁴) Angiotensin II (Amersham International), and rabbit angiotensin II (human) antiserum (Amersham International). The limit of detection of angiotensin II was 0.9 pmol/l, the interassay variability was 10% and the intraassay variability was 6%. Plasma aldosterone immunoreactivity was determined with the RSL [¹²⁵I] aldosterone kit (ICN Biomedicals, Inc., Costa Mesa, CA) with a limit of detection of 2.0 pmol/l and an interassay variability of 4% and in intraassay variability of 10%.

Immunohistochemistry

Paraffin-embedded lung cancer samples were identified from the patients participating in this study. Sections of the tumor tissues were incubated with guinea pig anti-sera to AVP and ANP (GAS 8103N and 8798N, respectively; Peninsula Laboratories, Belmont, CA) at 1:400 dilutions at room temperature for 30 minutes (30). Antibody-bridge complexes were then assembled using the biotinylated secondary antibody using the manufacturer's instructions (Vector Laboratories, Burlingame, CA). Immunostaining was then performed using 3-amino-9-ethylcarbazole as the chromogen. The slides were counter stained with Mayer's hematoxylin and observed for evidence of staining. The slides which revealed staining in the cytoplasm of cells with AVP and ANP antibody were considered positive. The sensitivity and specificity of the immunoreactions were verified with the use of human posterior pituitary as a source for AVP and the human atrial myocardium as a source for ANP. Serum from rabbits which have not been immunized were used as a source for nonspecific antibodies.

Data Analysis

The first set of analyses tested the association between the occurrence of hyponatremia and the plasma hormone levels. Both the initial ("0" minutes) and the median results of the seven samples taken from each patient were used in the analyses. The associations between plasma AVP, and both hyponatremia and tumor histology were analyzed using Fisher's exact test. The tests involving ANP, PRA, angiotensin II, and aldosterone were all Wilcoxon rank sum tests. Correlation between plasma ANP or AVP levels and urinary sodium were analyzed using the Spearman rank correlation method.

The second set of analyses was the test for trends over time in the plasma hormone levels using the seven serial samples taken from the patients. The direction of the change in hormone levels in each patient was assessed and analyzed using repeated measures analysis of variance and the Wilcoxon signed rank test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Population

One hundred forty-six patients with lung cancer were evaluated for inclusion and fifty patients with previously untreated lung cancer participated in this study between May 1989 and July 1992 (Table 1). Thirty-one of 61 (51%) previously untreated patients with small cell lung cancer seen at the Medicine Branch participated in the trial. Of the 30 who were not entered on study, 15 had hypertension or significant cardiovascular disease, eight declined the opportunity to participate, and seven required urgent therapy. Nineteen of 85 (22%) patients with non-small cell lung cancer evaluated at the Medicine Branch participated in the trial. Of the 66 not entered on study, 46 had received prior therapy, 10 refused to participate, and 10 had hypertension or significant cardiovascular disease. Eleven of the 50 (22%) patients with lung cancer participating in this study had hyponatremia, 10 of 31 (35%) with small cell lung cancer and one of 19 (5%) with non-small cell lung cancer (Table 1). The studies presented in the rest of this article deal with the 50 patients who participated in the trial.

Arginine Vasopressin Studies

Fifteen of the 50 patients with lung cancer had inappropriately elevated plasma AVP levels when correlated with serum osmolality (Figure 1). All 11 patients who presented with hyponatremia had inappropriately elevated levels of AVP. Four patients with normal serum sodium levels also have inappropriately elevated levels of AVP, 3 patients with small cell lung cancer and 1 with non-small cell lung cancer. As seen in Figure 1, the four patients with normal serum sodium values had AVP levels which were just outside of the normal range. Thirty-five patients had normal plasma levels of AVP, and none of these patients were hyponatremic. Elevated plasma AVP levels were highly correlated with the presence of hyponatremia (p < 0.00001). Association of evaluated plasma AVP levels with small cell lung cancer histology reached standard statistical significance (p = 0.026). There was a weak negative significant association between plasma AVP levels and urinary sodium levels (p = 0.05); patients with elevated AVP levels had low concentrations of sodium in their urine.

View larger version (34K): [in this window] [in a new window]   Figure 1.   Relationship between initial plasma AVP levels and initial plasma osmolality in patients with lung cancer. The shaded area represents the normal range as defined by Robertson and coworkers (7). Values to the left of the shaded areas represent inappropriately elevated levels of AVP. The value identified as 490 pmol/l is off the scale. Low sodium is defined as less than or equal to 130 mEq/l and normal sodium is values above those levels.

Atrial Natriuretic Peptide Studies

Although the median value of plasma ANP in patients with non-small cell lung cancer was slightly higher than for patients with small cell lung cancer, there was extensive overlap between the values and the differences did not reach standard statistical significance (Figure 2). Fourteen patients had elevated levels of ANP, including three who presented with small cell lung cancer and hyponatremia. The other 11 patients with elevated levels of ANP had normal serum sodium levels, 4 patients with small cell lung cancer and seven with non-small cell lung cancer. Thirty-six patients had normal plasma levels of ANP, and 8 of these patients were hyponatremic. There was no significant association between elevated plasma ANP levels and hyponatremia, either when initial plasma samples (p = 1.0) or median plasma values (p = 0.32) were analyzed. Similarly, there was no significant association between elevated plasma levels of ANP and tumor histology (p = 0.34 and p = 0.43 for initial and median values, respectively). In addition, there was also no association between elevated plasma ANP levels and either elevated plasma AVP levels (p = 1.0 and p = 0.35 for initial and median ANP values, respectively) or urinary sodium levels (p = 0.92 and p = 0.35 for initial and median values, respectively).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Individual and median values of baseline hormonal levels in patients with non-small cell lung cancer, small cell lung cancer with normal sodium, and small cell lung cancer and hyponatremia. The open circles and squares represent the patients with hyponatremia (sodium  130) while the closed circles and squares represent those patients with normal sodium. The ANP is depicted on a log scale because it is produced ectopically and is therefore present in the plasma over a wide range of concentrations.

Ectopic Production of Arginine Vasopressin and Atrial Natriuretic Peptide

Tumor cell lines were established from four patients participating in this study, three with small cell lung cancer (NCI-H2227, NCI-H2332, and NCI-H2679), and one with non-small cell lung cancer (NCI-H2562). The three patients with small cell lung cancer had normal serum sodium values at the start of the study (135-137 mEq/l). The patient with non-small cell lung cancer had a low serum sodium at the start of the study (127 mEq/l). Review of the pathology of this patient showed large cell carcinoma with neuroendocrine features (Figure 3). The tumor cell line NCI-H2562 was established from the biopsy specimen of the patient with non-small cell lung cancer and hyponatremia. The patient's serum sodium rose when the patient's tumor responded and fell when the patient relapsed (Figure 4), characteristic of an ectopic hormone caused syndrome. Reverse transcriptase-polymerase chain reaction showed none of these four expressed AVP mRNA (Figure 5A). In contrast, NCI-H2562 and NCI-H2679 expressed ANP mRNA (Figure 5B). NCI-H2562 had the highest amount of immunoreactive ANP detected in the cell pellet and NCI-H2679 the third highest (Table 2). The four lung cancer cell lines did not have significant amounts of detectable AVP immunoreactivity. Twenty-eight tumor samples were identified from 25 patients, which were adequate for immunohistochemical analysis. Eleven specimens were from patients with non-small cell lung cancer (one with hyponatremia) and 14 were from patients with small cell lung cancer (three with hyponatremia). Despite the presence of detectable AVP in the posterior pituitary and ANP in the atrial myocardium, there was no detectable peptide in any of the tumors specimens from the 25 patients (data not shown).

View larger version (165K): [in this window] [in a new window]   View larger version (128K): [in this window] [in a new window]   Figure 3.   Photomicrographs of a slide prepared from a right supraclavicular lymph node biopsy. (A) The morphology reveals a large cell carcinoma (hematoxylin and eosin, original magnification ×400). (B) Immunostaining for cytokeratin shows paranuclear-dot-reactivity supporting the diagnosis of a neuroendocrine tumor. An immunostain for neurofilaments showed immunoreactivity while chromogranin, leu-7 and bombesin immunostained were unreactive in the same specimens. (Immunoperoxidase stain for keratin, original magnification ×400.)

View larger version (13K): [in this window] [in a new window]   Figure 4.   Serum sodium values for patient with non-small cell lung cancer and hyponatremia. Day 1 refers to the day 66 Gy of chest radiotherapy was started for the patient. The values before 0 refer to the sodium levels prior to the initiation of treatment while the positive numbers refer to the number of years after starting treatment. The normal serum values for sodium are between 137 to 145 mEq/l represented by the dashed lines. The patient's tumor resolved with the chest radiotherapy. He relapsed in the liver and left upper quadrant 0.6 years after starting treatment, received 8 courses of etoposide cisplatin with a partial response. He relapsed again 1.3 yr after the start of treatment and additional salvage therapies did not reduce his cancer. He died from cancer 1.6 yr after starting treatment.

View larger version (48K): [in this window] [in a new window]   Figure 5.   RT-PCR analysis of mRNA from lung cancer cell lines. (A) The signal at 293 BP represents the amplified signal present in the cell line NCI-H711 previously shown to express AVP mRNA but not in any of the tumor cell lines from the patients participating in this study. (B) The signal at 236 PB represents the amplified signal represent in the cell line NCI-H1284 previously shown to express ANP mRNA and NCI-H2562 and 2679.

Plasma Renin Activity, Angiotensin II, and Aldosterone Studies

Thirty patients entered on this study had plasma renin activity, angiotensin II, and aldosterone levels measured. Seven patients had a single determination of their plasma AVP and ANP and did not participate in the saline infusion portion of the study or have their own hormones measured. The other 13 patients underwent the saline infusion and had their AVP and ANP measured every 15 min but did not have their other hormone levels determined. The plasma renin activity had similar median values and broad overlap between the three different patients groups (Figure 2). There was no significant association of PRA with the presence of hyponatremia (p = 0.67) or the type of histology (p = 0.90). The median angiotensin II levels were higher in the patients with small cell lung cancer although there was overlap between the three different patient groups and the differences did not achieve standard statistical significance. Four patients had elevated levels of angiotensin II at presentation; one normonatremic patient with small cell lung cancer and three normonatremic patients with non-small cell lung cancer. There was no correlation between plasma angiotensin II levels, both baseline and median, and hyponatremia (p = 0.47 and p = 0.63, respectively). Similarly there was no relationship between plasma angiotensin II levels and lung cancer histology. One patient had an elevated level of aldosterone at the start of the study. Although the median aldosterone levels were higher in the patients with small cell lung cancer, there was overlap between the three different patient groups and the differences did not achieve standard statistical significance. Hence there was no significant correlation between plasma aldosterone levels, either baseline or median, and the occurrence of hyponatremia (p = 0.12 and p = 0.18, respectively). The plasma ANP was correlated with the plasma renin activity and aldosterone because of their inverse relationship in normal sodium homeostatis. The initial ANP level was not inversely correlated with the plasma renin activity and aldosterone by the Spearman rank correlation coefficient (rho = 0.04, p = 0.83 and rho = 0.01, p = 0.95). Because of the lack of correlation between these hormones and the occurrence of hyponatremia, the final 13 patients did not have their plasma levels assayed.

Physiologic Response to Postural Change and Infusion of Saline

Forty-two patients had their blood pressure determined in the supine and standing position before their saline infusion. Eleven had a drop in their systolic or diastolic pressure of 10 mm Hg or more going from the supine to the standing position. The 11 patients with postural hypotension had a median ANP of 37.3 pmol/l (range 1.7-208) compared with 11.5 (range 1.8-147) for the patients who did not have postural hypotension. This difference was not statistically significant (p = 0.28). In contrast, the 11 patients with postural hypotension had a median AVP of 1.2 pmol/l (range 0-490) compared to 0.2 (range 0-23.6) for the patients who did not have postural hypotension. This difference was statistically significant (p = 0.012).

The response to 500 ml of intravenous saline administration were assessed in three groups of patients, patients with small cell lung cancer and hyponatremia (7), patients with small cell lung cancer and normal serum sodium (15), and patients with non-small cell lung cancer (17) (Figure 6). The median values for each group and all individual values were examined. Physiologic responses to intravenous saline administration were detectable. There was a steady fall in plasma aldosterone levels during saline administration from 15 to 60 min, with a sudden rise at the 75- and 90-min marks. The last four changes are all significant (p < 0.01). Similarly the angiotensin II levels tended to decrease at the 15 min mark and remain low throughout the remainder of the study (p < 0.05). Plasma AVP levels and PRA in all three patient groups were largely unchanged during and after the saline infusion. There were no significant differences or trends in plasma ANP levels among the three patient groups.

View larger version (30K): [in this window] [in a new window]   Figure 6.   The effects of saline load on arginine vasopressin, atrial natriuretic peptide, plasma renin activity, angiotensin II, and aldosterone. The bar at the top of the figure represents 500 ml of saline administered over 45 min. The circles and lines represent the median values for the patients and the bars represent the 25th and 75th percentile values.

The concentration of urinary sodium increased in all three groups (Figure 7). There was an association between the increase in urinary sodium and baseline ANP levels (rho = 0.45, p = 0.0045). The relationship was more evident in the eight patients with hyponatremia (rho = 0.96, p = 0.0007) than in the 31 patients with normal sodium (rho = 0.34, p = 0.064).

View larger version (13K): [in this window] [in a new window]   Figure 7.   Relationship between initial plasma ANP levels and sodium excretion after a saline load in lung cancer patients. This represents the percentage increase in the sodium concentration of the urine before and 90 min after an infusion of 500 ml of saline for patients with non-small cell lung cancer and small cell lung cancer with normal sodium and with hyponatremia.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this prospective study, 10 of 31 (32%) patients with small cell lung cancer had hyponatremia. This percentage appears to be higher than previous retrospective studies which report approximately 15% of patients presenting with hyponatremia (3) and confirms that hyponatremia is uncommon in patients with non-small cell lung cancer. This study once again clearly shows the close association between elevated AVP and hyponatremia in patients with small cell lung cancer.

As stated before, a third of the patients with small cell lung cancer and hyponatremia have no evidence of ectopic production of AVP in either their tumor or tumor cell line (4, 6, 8). These patients have evidence of ectopic production of atrial natriuretic peptide. Our patient with non-small cell lung cancer and hyponatremia had a tumor cell line established (NCI-H2562) which ectopically produced the hormone, ANP. This tumor cell line had evidence of ANP mRNA expression and immunoreactivity (39 pmol/g) without evidence of AVP mRNA expression or significant immunoreactivity. The ANP plasma level in this patient was 4-12 pmol/l, which is not particularly high compared with the median plasma ANP levels of 17 pmol/l (range 2-137) reported in 69 patients with small cell lung cancer (6).

There has been other recent evidence which supports the role of ANP playing a role in hyponatremia in lung cancer. Campling and coworkers presented data on a patient with small cell lung cancer who presented with hyponatremia (serum sodium of 127 mEq/l) (6). This patient had her tumor resected and a tumor cell line was established (LD-T). Analysis of this tumor cell line showed 242 pg/mg of soluble protein (79 pmol/g) and undetectable AVP. Her serum sodium returned to normal after surgical resection of her small cell lung cancer. The plasma levels of ANP were not available from this patient. In our study and the study by Campling and coworkers (6), ANP production in patients with lung cancers has been documented by mRNA expression, ANP immunoreactivity in the tumor cell line cytosol and supernatant, and the hyponatremia has resolved after surgical resection, chest radiotherapy, and systemic chemotherapy.

Despite this evidence of ectopic production of ANP and hyponatremia in these two patients, there has been no consistent association between elevated ANP and hyponatremia in patients with lung cancer. Two studies of ANP in the plasma or tumor cell lines showed no direct relationship between high levels of ANP and hyponatremia in patients with lung cancer (4, 6). The plasma concentration of ANP increases with increasing amounts of sodium intake potentially explaining some of the wide variation in plasma ANP levels in patients with lung cancer (14, 15). In addition, oral intake of sodium may offset potential urinary losses caused by atrial natriuretic peptide.

This study has provided some new information about the physiologic interaction between AVP, ANP and the renin- angiotensin-aldosterone (RAA) system. There was no association between the plasma levels of ANP and renin, angiotensin II, and aldosterone. The RAA system, under normal circumstances, is activated to promote sodium conservation under conditions of sodium deficit, volume contraction, or reduction in renal perfusion pressure (1, 2, 15). We had postulated that elevated levels of ANP could decrease the plasma levels of plasma renin activity, angiotensin II and aldosterone. The plasma renin activity were similar in all three groups while angiotensin II and aldosterone levels were higher in the patients with small cell lung cancer than non-small cell lung cancer and none of the values were particularly low. This suggests that the higher levels of ANP in some of the patients ectopically producing ANP did not cause lower plasma hormones in the renin-angiotensin-aldosterone system. Therefore, if inappropriately produced ANP is postulated to be present in some of these patients, it does not appear to decrease the amounts of the renin-angiotensin-aldosterone present in these patients. There is no evidence for ANP suppressing the renin-angiotensin-aldosterone system leading to hyponatremia and a negative sodium balance.

Hormonal responses to sodium loading, both with increased oral sodium ingestion and intravenous saline, have been well documented in the literature (31). Some of the characteristic changes that occur after administration of oral or intravenous saline were observed in these patients with lung cancer including reduction in plasma aldosterone and an increase in urinary sodium excretion showing that the saline load induced a physiologic response. The decrease in the plasma aldosterone with the administration of 500 ml of saline shows that aldosterone is able to response physiologically to a saline challenge despite the presence of ectopic hormones in some of these lung cancer patients.

There is some evidence that ectopic production of ANP could contribute to hyponatremia by causing a natriuresis, negative sodium balance, and nonosmotic release of AVP because of decreased intravascular volume. Patients increased their urinary sodium concentration proportional to their initial plasma level of ANP (p = 0.0045) after receiving intravenous saline. This was most prominent in the patients with hyponatremia (p = 0.0007). There was also a trend for patients with orthostatic decreases in blood pressure and/or increases in the pulse to have higher levels of ANP although it did not achieve standard statistical significance. These patients with orthostatic changes did have higher levels of AVP (p = 0.012) consistent with nonosmotic release of AVP because of decreased intravascular volume. This suggests that these patients had volume depletion rather than volume overload. Another potential explanation of these findings are lesions in afferent nerves from peripheral baroreceptors inappropriately stimulating the release of AVP from the hypothalamus (1, 2, 7).

The contribution of ectopically produced AVP and ANP to the development of hyponatremia in patients with lung cancer will be able to be investigated more thoroughly with newly developed antagonists. A recently added important pharmacologic agent for studying the effects of AVP are the orally active, nonpeptide AVP antagonists of the V₂ receptor (34). These agents will allow investigators to administer antagonists to patients with hyponatremia of malignancy. Those with hyponatremia of malignancy mediated by AVP through the V₂ receptor will likely correct their hyponatremia when treated with an AVP antagonist. Patients with hyponatremia mediated by peptides other than AVP (ANP is a candidate peptide) will not likely correct their hyponatremia. The role of ectopic ANP production in patients with lung cancer will need to be established by the administration of a specific ANP antagonist when available for clinical investigation in humans. If patients with hyponatremia of malignancy have their low sodium caused by ectopic production of ANP mediated through the ANP receptors, these patients will likely correct their hyponatremia when treated with an ANP antagonist.

Bartter's and Schwartz's original description of SIADH, proposed three potential factors which could explain sodium loss with SIADH (35). These included: (1) a factor causing suppression of aldosterone secretion resulting from an increase of extracellular fluid volume; (2) a factor causing an increase in the filtered load of sodium resulting from an increase in the glomerular filtration rate; and (3) a factor causing the suppression of tubular reabsorption of sodium in response to expansion of the extracellular fluid volume. The data from this study do not support ANP as the first potential factor. There is no evidence of suppression of aldosterone secretion in these patients and they are still able to decrease their plasma levels of aldosterone in response to a saline infusion. The properties of ANP are still consistent with the other two of Bartter's and Schwartz's proposed factors. Atrial natriuretic peptide increases the glomerular filtration rate, and inhibits sodium reabsorption into the renal tubules (14, 15). Thus ectopically produced ANP may play a role in the etiology of hyponatremia associated with lung cancer, either alone, or in concert with ectopic production of AVP.