Diagnosis of central diabetes insipidus using a vasopressin radioimmunoassay during hypertonic saline infusion

Abstract

Central diabetes insipidus (CDI) is characterized by polyuria and polydipsia caused by impairment of arginine vasopressin (AVP) secretion. In this study, we evaluated plasma AVP concentrations during a hypertonic saline infusion test using a new AVP radioimmunoassay (RIA) which is now available in Japan. Thirteen control subjects, mostly with hypothalamo-pituitary disease but without CDI, and 13 patients with CDI were enrolled in the study. Whether or not subjects had CDI was determined based on the totality of clinical data, which included urine volumes and osmolality. Regression analysis of plasma AVP and serum Na concentrations revealed that the gradient was significantly lower in the CDI group than in the control group. The area under the receiver-operating-characteristic (ROC) curve was 0.99, and the <0.1 gradient cut-off values for the simple regression line to distinguish CDI from control had a 100% sensitivity and a 77% specificity. The ROC analysis with estimated plasma AVP concentrations at a serum Na concentration of 149 mEq/L showed that the area under the ROC curve was 1.0 and the <1.0 pg/mL cut-off values of plasma AVP had a 99% sensitivity and a 95% specificity. We conclude that measurement of AVP by RIA during a hypertonic saline infusion test can differentiate patients with CDI from those without CDI with a high degree of accuracy. Further investigation is required to confirm whether the cut-off values shown in this study are also applicable to a diagnosis of partial CDI or a differential diagnosis between CDI and primary polydipsia.

CENTRAL DIABETES INSIPIDUS (CDI) is a disease characterized by polyuria and polydipsia caused by a deficiency of arginine vasopressin (AVP), an antidiuretic hormone synthesized in the hypothalamus and released from the posterior pituitary into the systemic circulation. Relatively low plasma AVP concentrations at high plasma osmolality or serum sodium (Na) concentrations in patients are theoretically indicative of a diagnosis of CDI. However, validated assays for plasma AVP are unavailable in most countries, where a water deprivation test has been used for the diagnosis [1-3]. In a water deprivation test, patients are requested to tolerate thirst for at least several hours, and those with complete diabetes insipidus could become severely dehydrated. In addition, previous reports of the 70–78% accuracy of a diagnosis based on a water deprivation test highlight a limitation [4, 5]. Recently, the measurement of copeptin, a peptide which is cleaved from AVP precursors and released into the circulation in equimolar amounts with AVP, has been challenged [4-8], and a hypertonic saline infusion test together with copeptin measurement reportedly improved the diagnostic accuracy up to 96% [5]. However, as copeptin is a surrogate marker of AVP [6], the importance of establishing a system by which plasma AVP concentrations are assessed accurately cannot be overstated.

In Japan, CDI had been diagnosed based on plasma AVP concentrations measured with a sensitive assay for AVP (Mitsubishi Petrochemical Co. Ltd., Tokyo, Japan) [9, 10], but the assay kit has been unavailable since 2013. The purpose of this study was to examine whether increased plasma AVP concentrations during a hypertonic saline infusion test can be detected in control subjects with a new assay [11], and if detectable, to examine whether CDI can be diagnosed accurately using a hypertonic saline infusion test together with measurement of plasma AVP concentrations.

Materials and Methods

Patients

Thirteen subjects (6 males, 7 females) without CDI (control group) and 13 patients (7 males, 6 females) with CDI were enrolled in this study. Average ages were 57.9 ± 4.2 years in the control group and 50.1 ± 3.2 years in the CDI group. Subjects were judged not to have CDI if (1) urine volumes were less than 3,000 mL/day, (2) urine osmolality was higher than 300 mOsm/kg under euhydrated conditions or higher than 600 mOsm/kg after a water deprivation test, and (3) there existed a hyperintense signal on a T1-weighted pituitary MRI. The diagnosis of CDI was made based on the totality of clinical data which included urine volumes, urine osmolality, absence of a hyperintense signal on a T1-weighted pituitary MRI, and responses to desmopressin. All enrollees were examined at Nagoya University Hospital from March, 2015 to August, 2019. The study was approved by an appropriate institutional review board (IRB) at Nagoya University, and written informed consent was obtained from all the participants. The study was registered to the University Hospital Medical Information Network (UMIN), registry number UMIN000020112.

Hypertonic saline infusion test

During the hypertonic saline infusion test, patients rested in a supine position on a bed for 30 min before the infusion was initiated at 9:00 AM. Hypertonic (5%) saline was infused at a rate of 0.05 mL/kg/min for 120 min. Blood samples were collected in tubes containing ethylenediaminetetraacetic acid disodium salt 2-hydrate before and 30, 60, 90, and 120 min after initiation of the infusion. Collected samples were immediately kept on ice and plasma was separated by centrifugation. Separated plasma was stored at −20℃ in the refrigerator and subjected to the assay within 1 month following sample collection.

Water deprivation test

Fluid restriction for 6.5 h was started at 9:00 AM. Every 2 h, urine was collected for volume and osmolality measurements. Body weight and vital signs were also monitored every 2 h.

Changes in plasma AVP concentrations after DDAVP treatment

To determine whether AVP assays exhibit any cross-reactivity with 1-deamino-8-D-arginine vasopressin (DDAVP)—an analog of AVP which has been used for the treatment of CDI—8 patients with CDI were administered desmopressin nasally (2.5 μg, n = 4) or orally (60 μg, n = 4). Plasma samples were collected before and 1 h after administration of DDAVP for AVP assays.

Plasma AVP measurement

Plasma AVP concentrations during hypertonic saline infusion tests were measured with a RIA kit (YAMASA Shoyu Corporation, Choshi, Japan) using a primary rabbit polyclonal antibody. In this assay, the extraction of AVP from plasma was carried out using a cold ethanol method; the detection limit of this assay is 0.4 pg/mL [11]. Cross reactivities with arginine vasotocin, lysine vasopressin, and DDAVP have been reported 0.09%, 0.001%, and <0.001%, respectively [11]. Plasma AVP concentrations before and 2 h after administration of DDAVP were measured using 2 different AVP RIA kits, one from YAMASA and the other from LSI Medience (Tokyo, Japan). The reported detection limit and cross reactivity with DDAVP of the AVP RIA kit from LSI Medience are 0.8 pg/mL and 0.88%, respectively, according to the manufacturer’s instructions.

Statistical analysis

Serum Na and plasma AVP concentrations were examined using regression analysis. For each subject, a simple regression line was calculated to represent the correlation between serum Na and plasma AVP concentrations during a hypertonic saline infusion test. The estimated gradients of the regression line (regression coefficients) were compared between the control and CDI groups. Based on the estimated linear lines, plasma AVP concentrations at serum Na concentrations of 140–153 mEq/L were also estimated and compared between the control and CDI groups. The receiver-operator characteristics (ROC) curve and the area under the curve (AUC) with its 95% confidence interval, calculated by the DeLong method, were applied to evaluate diagnostic accuracy. The analysis was performed using SAS 9.4 version (SAS Institute Inc., Cary, NC, USA) and R version 3.6.0 (R Development Core Team).

To examine whether plasma AVP concentrations measured using 2 AVP kits increased after desmopressin treatment, a paired-t test was performed. All continuous data were reported as means ± SE.

Results

Patients

In the control group, 10 out of 13 patients (76.9%) had hypothalamo-pituitary diseases (acromegaly, n = 8; adult GH deficiency, n = 1; and craniopharyngioma n = 1); the treatments given to those patients and tumor sizes are shown in Table 1. Hyperintense signals on T1-weighted pituitary MRIs were detected in all 10 cases. Two patients (Cases 6 and 12) in the control group were admitted to our hospital after reporting symptoms of polyuria. However, the urine volumes were <2 L/day [Case 6: 1,800 mL/day (44.1 mL/kg/day); Case 12: 1,500 mL/day (19.9 mL/kg/day)]. Urine osmolalities increased to >600 mOsm/kg (Case 6: 626 mOsm/kg; Case 12: 616 mOsm/kg) and urine volumes decreased during a water deprivation test in both cases. The patient in Case 2 was admitted to our hospital after reporting symptoms of fatigue and thirst. Examinations showed that the urine volume was <2 L/day [1,300 mL/day (30.7 mL/kg/day)], and that the urine osmolality was 496 mOsm/kg under a euhydrated condition. Hyperintense signals on T1-weighted pituitary MRIs were also confirmed in these 3 patients. Thus, these 3 cases in the control group were judged normal in terms of the hypothalamo-neurohypophysial system. Average urine volumes in the control group were 1,529 ± 117 mL/day (21.4 ± 3.1 mL/kg/day) (n = 13), and the average urine osmolarity under euhydrated conditions was 527 ± 47 mOsm/kg (n = 13) (Table 1).

Table 1 Characteristics of patients in the control group

Case No.	Gender	Age (yr)	Diagnosis	Urine volume		Urine osmolality (mOsm/kg)	Timing of evaluation	Tumor size (mm)
				(mL/day)	(mL/kg/day)
1	F	38	Cranio-pharyngioma	638	11.3	468	before TSS	13
2	F	26	Normal	1,300	30.7	496	—	—
3	M	45	Acromegaly	1,400	20.2	607	after TSS	n.d.
4	F	76	Acromegaly	2,150	35.6	484	after TSS	13
5	M	63	Acromegaly	1,850	21.0	498	after TSS	7
6	F	63	Normal	1,800	44.1	311	—	—
7	F	72	Acromegaly	1,145	24.2	419	before TSS	15
8	F	57	Adult GH deficiency	2,110	36.1	575	after TSS and radiation	5
9	M	53	Acromegaly	1,655	19.9	950	—	13
10	M	49	Acromegaly	1,300	14.9	639	after TSS	n.d.
11	M	71	Acromegaly	1,250	20.4	618	after TSS	n.d.
12	F	75	Normal	1,500	25.0	290*	—	—
13	M	65	Acromegaly	1,780	18.1	531	after TSS	10

F, female; M, male; n.d., not detected; TSS, transsphenoidal surgery. *, Urine osmolality increased to >600 mOsm/kg during a water deprivation test.

In the CDI group, 6 cases were previously diagnosed with CDI and had been treated with DDAVP, as shown in Table 2. Symptoms of polydipsia and polyuria resolved in these patients with DDAVP treatment. During the period of this study, CDI was newly diagnosed (yr = 0) in the remaining 7 cases that reported symptoms of polyuria and polydipsia. The average of daily urine volumes in the CDI group was 5,905 ± 773 mL (79.4 ± 14.7 mL/kg/day) (n = 11), and urine osmolality was 130 ± 10 mOsm/kg (n = 8) before DDAVP administration. Urine volumes were reduced and urine osmolality increased by DDAVP administration in all of the patients newly diagnosed with CDI. On the T1 MRIs of the pituitary, the high-intensity signal was absent or reduced in all of the patients with CDI. The diagnosis of CDI was thus made based on all the available clinical data, as in a previous study [5]. Four cases were accompanied by other endocrinological diseases such as secondary adrenal failure and hypothyroidism. In these patients, hydrocortisone and thyroid hormone were adequately replaced before the hypertonic saline infusion tests.

Table 2 Characteristics of patients in the CDI group

Case No.	Gender	Age (yr)	Time after diagnosis (yr)	Urine volume		Urine osmolality (mOsm/kg)	Etiology	Route of DDAVP administration (μg/day)	Coexisting diseases
				(mL/day)	(mL/kg/day)
1	M	42	0	9,000	124.0	111	Idiopathic	oral (180)
2	M	46	0	5,600	87.6	103	Idiopathic	oral (120)
3	F	62	0	8,060	155.0	n.d.	Multiple vascular granuloma	oral (120)
4	M	53	1	5,000	47.5	n.d.	Pituitary tumor (apoplexy)	oral (120)
5	F	40	0	4,000	89.1	n.d.	Pituitary tumor	nasal (2.5)
6	F	38	4	8,400	166.7	n.d.	Craniopharyngioma (postoperative)	oral (240)	ACTH deficiency, Hypothyroidism
7	M	56	3	n.d.	n.d.	134	Pituitary tumor (postoperative)	oral (60)	ACTH deficiency, GH deficiency, Hypogonadism
8	F	32	0	5,600	107.7	126	Pituitary tumor (apoplexy)	oral (60)
9	M	47	2	3,058	41.1	n.d.	IgG4-related hypophysitis	oral (180)	ACTH deficiency, GH deficiency, Hypogonadism
10	F	45	9	10,000	108.0	n.d.	Idiopathic	oral (120)	Hashimoto’s thyroiditis
11	F	53	0	3,500	53.0	159	Pituitary tumor (postoperative)	oral (60)
12	M	64	27	n.d.	n.d.	n.d.	Idiopathic	nasal (5)
13	M	73	0	2,740	52.7	n.d.	IgG4-related hypophysitis	oral (60)

M, male; F, female; n.d.: no data available.

Statistical analysis to establish a threshold to diagnose CDI

Serum Na and plasma AVP concentrations during hypertonic saline infusion test were shown in Fig. 1 and Table 3. During the hypertonic saline infusion, 2 patients reported slight pain at the injection site and a headache, but both events were tolerable and no other adverse events were reported.

Fig. 1

Serum sodium and plasma AVP concentrations during hypertonic saline infusion tests.

A, Control group (n = 13). B, CDI group (n = 13). C, Comparison between control and CDI groups.

The 95% prediction intervals for plasma AVP concentrations during a hypertonic saline test are shown by dotted lines in each group.

Table 3 Serum sodium and plasma AVP concentrations during hypertonic saline infusion in the control and the CDI groups

	Control	CDI
Na (mEq/L) (0 min)	141 ± 0.4	143 ± 1.0
Na (mEq/L) (30 min)	144 ± 0.5	147 ± 1.2
Na (mEq/L) (60 min)	146 ± 0.6	150 ± 1.1
Na (mEq/L) (90 min)	148 ± 0.7	152 ± 1.0
Na (mEq/L) (120 min)	149 ± 0.6	153 ± 1.0
AVP (pg/mL) (0 min)	0.8 ± 0.08	0.6 ± 0.06
AVP (pg/mL) (30 min)	1.2 ± 0.11	0.6 ± 0.05
AVP (pg/mL) (60 min)	1.6 ± 0.17	0.6 ± 0.04
AVP (pg/mL) (90 min)	2.0 ± 0.20	0.7 ± 0.08
AVP (pg/mL) (120 min)	2.9 ± 0.34	0.8 ± 0.08

Data are means ± SE.

To establish the diagnostic threshold based on the plasma AVP and serum Na concentrations during the hypertonic saline infusion test, we calculated the single regression line for each subject (indexed by i) as follows.

(AVP)_i = αi + βi × (Na)_i β: regression coefficient

The 95% prediction intervals for the plasma AVP concentrations were constructed based on a normal linear mixed model with a compound symmetry variance-covariance structure (Fig. 1). This model was fit to repeated measures of each group separately to cope with a large difference in variance between the control and CDI groups. We restricted the model in the control group to have the same estimate of the plasma AVP concentration with that of the CDI group at a serum Na concentration of 140 mEq/L, given that the threshold of plasma osmolality for AVP release has been reported to be 280–284 mOsm/kg [12].

The average gradient of the regression lines (average regression coefficient) in the control group was 0.24 ± 0.04 (range, 0.07 to 0.45), while that in the CDI group was 0.015 ± 0.007 (range, −0.020 to 0.090) (Fig. 2A). The gradients were significantly higher in the control group than in the CDI group. The AUC of ROC was 0.99 [95% confidence interval (95% CI): 0.96–1.00)] (Fig. 2B). The <0.1 gradient cut-off values for the simple regression line used to distinguish CDI from control had a 100% sensitivity and a 77% specificity.

Fig. 2

Comparison of gradients of a single regression line during hypertonic saline infusion tests.

A. Gradients (white: control, black: CDI) and 95% confidence interval.

B. Receiver-operating-characteristic (ROC) curve of the gradient used for the diagnosis of CDI. The area under the curve (AUC) of the ROC curve was 0.99 (95% confidence interval: 0.96–1.00).

C. Distribution of estimated plasma AVP concentrations based on the linear approximate equation at a serum Na concentration of 149 mEq/L.

D. The ROC curve of the estimated plasma AVP concentrations based on the linear approximate equation at a serum Na concentration of 149 mEq/L. The AUC of the ROC curve was 1.00.

The ROC analysis with estimated plasma AVP concentrations based on the linear line revealed that the AUC was 1.0 at serum Na concentrations of 146–153 mEq/L (Table 4). The histogram of plasma AVP concentrations at a serum Na concentration of 149 mEq/L (mean values after hypertonic saline injection in the control group) and ROC analysis are shown in Fig. 2C and D, respectively. Because of a complete separation of the plasma AVP concentrations at a serum Na concentration of 149 mEq/L between the two groups, a model-based ROC analysis was performed to estimate a sensitivity and a specificity at a particular cut-off point. Specifically, the <1.0 pg/mL cut-off values of plasma AVP concentrations have a 99% sensitivity and a 95% specificity, based on the assumption that the plasma AVP concentrations are normally distributed in each group and that the prevalence of CDI patients is 0.5 among those subjected to the test.

Table 4 Estimated plasma AVP concentrations at serum Na concentrations of 140–153 mEq/L based on the linear approximate equation

Na (mEq/L)	AVP (pg/mL) in Control	AVP (pg/mL) in CDI	AUC of ROC	95% CI
140	0.3 ± 0.17	0.4 ± 0.08	0.503	0.246–0.760
141	0.6 ± 0.14	0.5 ± 0.07	0.604	0.353–0.854
142	0.8 ± 0.12	0.5 ± 0.07	0.716	0.494–0.938
143	1.1 ± 0.11	0.5 ± 0.06	0.888	0.763–1
144	1.3 ± 0.12	0.5 ± 0.06	0.976	0.926–1
145	1.5 ± 0.13	0.5 ± 0.05	0.994	0.978–1
146	1.8 ± 0.16	0.5 ± 0.05	1.0
147	2.0 ± 0.18	0.5 ± 0.05	1.0
148	2.3 ± 0.22	0.6 ± 0.05	1.0
149	2.5 ± 0.25	0.6 ± 0.05	1.0
150	2.7 ± 0.29	0.6 ± 0.05	1.0
151	3.0 ± 0.32	0.6 ± 0.05	1.0
152	3.2 ± 0.36	0.6 ± 0.05	1.0
153	3.5 ± 0.40	0.6 ± 0.06	1.0

Data are means ± SE.

Changes in plasma AVP concentrations after DDAVP treatment

Although the plasma AVP concentrations were increased after DDAVP treatment when measured using the LSI Medience AVP-RIA kit (before: 1.1 ± 0.17 pg/mL; after: 2.4 ± 0.28 pg/mL, n = 8, p < 0.05), there were no significant differences in the values before and after DDAVP treatment when measured using the YAMASA AVP-RIA kit (before: 0.7 ± 0.21 pg/mL; after: 0.7 ± 0.15 pg/mL, n = 8), indicating no cross-reactivity with DDAVP.

Discussion

In this study, we evaluated plasma AVP concentrations during a hypertonic saline infusion test using an AVP RIA that is clinically available in Japan. Our data showed significant differences in the response of AVP release between the control and the CDI groups, making it possible to differentiate patients with CDI from control subjects with the hypertonic saline infusion test.

A water deprivation test is used to indirectly diagnose patients with CDI in many countries. However, the accuracy of water deprivation tests for the diagnoses of CDI is reportedly 70–78% [4, 5]. In addition, water deprivation requires patients with CDI to abstain from drinking water for at least several hours, leading to a comparatively more severe burden for CDI patients than a hypertonic saline infusion test [3].

Measurement of plasma AVP concentrations is technically difficult [13-17] and requires extraction of AVP from plasma. In the AVP RIA employed in this study, a cold ethanol method was used for extraction [11]. The AVP extraction rate is reportedly 0.74% and the lower limit of measurable plasma AVP is 0.4 pg/mL using the ethanol method [11]. Both the intra- and inter-assay (n = 5) coefficients of variation were <8% [11]. This study showed that the plasma AVP concentrations after a hypertonic saline injection, measured using the RIA, were high enough in the control group to differentiate patients with CDI from those without CDI.

In this study, we used single regression lines with serum Na and plasma AVP concentrations, which enabled us to evaluate the AVP secretory response to increasing serum Na concentrations. Our analysis demonstrated that the cut-off values of the <0.1 gradient and <1.0 pg/mL estimated plasma AVP concentrations at a serum Na concentration of 149 mEq/L had high sensitivities and specificities, indicating that CDI could be diagnosed using these indexes in most cases. Furthermore, our data clearly demonstrated that the YAMASA AVP-RIA had no cross-reactivity with DDAVP, indicating that it is possible to measure plasma AVP concentrations in patients with CDI, even when they have been treated with DDAVP.

Copeptin is secreted into the blood stream in equimolar amounts with AVP and remains stable for several days at room temperature in serum or plasma [7, 18-21]. Immunoassays for the detection of copeptin have been developed and recent studies support its clinical usefulness in the differential diagnosis of CDI and primary polydipsia during a hypertonic saline infusion test [5, 8]. However, it has yet to be determined whether copeptin, given its longer half-life in plasma compared with AVP, could be a reliable surrogate marker of AVP in patients with pathogenic conditions such as renal failure and cirrhosis [22]. To address these concerns, it is necessary to compare the concentrations of copeptin with those of plasma AVP simultaneously using a validated assay for each of them.

The first limitation of our study is the small sample size. Although studies with more participants should be undertaken in the future, it is notable that our data showed high sensitivity and specificity, despite the small numbers of participants. The second limitation is that the control group consisted mostly of patients with some hypothalamo-pituitary diseases, as the IRB at our university did not permit the inclusion of healthy individuals in the study. The IRB’s decision was based on the premise that use of a hypertonic saline infusion test should be limited to subjects who may potentially have CDI or the likelihood of CDI onset in the future, so that the data would be beneficial to the study participants. Although the clinical data of the subjects in the control group did not meet the criteria for CDI diagnosis, we cannot exclude the possibility that subjects in the control group had a partial deficit in the release of AVP. If this is the case, patients with partial CDI might be diagnosed as normal based on the cut-off values presented in this study. The third limitation is related to the non-inclusion of patients with primary polydipsia in this study. However, it has been shown that the increase in plasma AVP concentration in patients with primary polydipsia is as high as in healthy controls [23], although confirmation via the assay employed in this study is necessary.

In conclusion, our data provided cut-off values for the diagnosis of patients with CDI based on serum Na and plasma AVP concentrations during hypertonic saline infusion tests, measured using an AVP RIA kit that is now available in Japan. Further investigation is required to confirm whether the cut-off values shown in this study are also applicable to the diagnosis of partial CDI or a differential diagnosis between CDI and primary polydipsia.

Acknowledgments

None.

Disclosure

Financial support was provided by YAMASA Shoyu Corporation, which had no other role in the study.

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