Abstract
Background: Plasma free metanephrines are a more reliable analyte to measure than catecholamines for the biochemical diagnosis of pheochromocytomas. We hypothesized that the long persistence of total (sulfate-conjugated plus free) metanephrines in the blood might have a significant diagnostic value.
Methods: We measured plasma concentrations of catecholamines and total metanephrines (sulfate-conjugated plus free forms) by HPLC with amperometric detection, and neuropeptide Y (NPY) by an amplified ELISA in seven patients before and after removal of their pheochromocytomas. The results for catecholamine, total metanephrines, and NPY in each patient were analyzed for up to 120 min, starting from the time of tumor vessel clamping. The persistence of analytes was quantified as the area under the concentration–time curve over 120 min.
Results: On the basis of the upper reference limit for each variable, plasma free norepinephrine (NE) and epinephrine (E) concentrations were increased preoperatively in at least one sample in seven and six patients, respectively. Total normetanephrine (NMN) and metanephrine (MN) were increased in all samples in seven and six patients, respectively. NPY was increased 2- to 465-fold. After removal of the tumor, MN and NMN showed a higher average relative increase above the upper limit of the reference interval than NE and E (P = 0.05), whereas NPY was intermediate. The persistence of increased values was significantly shorter for catecholamines than for metanephrines. The half-life estimated by nonlinear regression was 12.3 ± 7.8 min for NPY. Significant correlations were observed among NE, E, NMN, MN, and NPY concentrations, but parent markers (E and MN or NE and NMN) did not appear significantly intercorrelated.
Conclusions: A larger increase and a longer persistence of total metanephrines (reflecting predominantly sulfo-conjugated metanephrines) than catecholamines and NPY in plasma may contribute to their greater diagnostic accuracy in pheochromocytoma.
Pheochromocytoma is a rare and potentially lethal tumor of chromaffin cells that secretes catecholamines directly into the circulation, producing episodes of hypertension with palpitations, severe headaches, and sweating (symptomatic triad) (1)(2). Traditional biochemical tests have relied on the determination of the 24-h urinary excretion of free catecholamines [norepinephrine (NE)1 and epinephrine (E)] or of metanephrines [normetanephrine (NMN) and metanephrine (MN)], their metabolic products, as the most clinically sensitive and specific biochemical tests for the detection of pheochromocytoma (2)(3)(4). Nevertheless, because of problems of incompleteness and inconvenience (acidification with hydrochloric acid) associated with 24-h urine collections, clinicians have long sought a diagnostic test based on sampling of venous blood. Plasma E and NE measurements suffer from problems of sample timing because of the intermittent characteristics of tumor secretion and instability, which generate false-negative results (4)(5). In contrast, emotional stress or other cardiovascular pathologic conditions, such as heart failure, can produce abnormally high catecholamine concentrations, causing false-positive results (6)(7). The measurement of plasma MN and NMN has been shown to be more clinically sensitive than urinary free catecholamines and metanephrines for the detection of pheochromocytoma (8)(9)(10)(11).
Membrane-bound catechol-O-methyltransferase has a higher affinity for catecholamines than does the soluble enzyme present in other tissues. Because membrane-bound catechol-O-methyltransferase is expressed preferentially by the adrenals (12), metanephrines are produced in relatively large amounts by healthy adrenal tissue (13). Thus, 90% of MN and 24–40% of NMN in plasma originate from the adrenal glands. As a result, plasma NMN has a higher sensitivity than does NE for detecting pheochromocytoma (13)(14). In addition to metabolic considerations, we also observed previously that total concentrations of metanephrines were normalized 5–12 days after removal of the pheochromocytoma (9). However, in contrast to the free metanephrines determined by other laboratories, our measurements of metanephrines were performed after acid hydrolysis, which led to deconjugation of NMN sulfate and MN sulfate to free NMN and MN. Importantly, plasma concentrations of free metanephrines are <5% of the concentrations of the conjugated metanephrines. Therefore, we hypothesized that the high diagnostic sensitivity of plasma total metanephrines is not only attributable to the ability of the tumor to O-methylate catecholamines, but also to a longer half-life of the sulfate-conjugated MN and NMN than free catecholamines.
Neuropeptide Y (NPY), like NE, is also a neurotransmitter released from the sympathetic nerve endings that causes vasoconstriction (15). In addition, NPY is produced by the healthy adrenal gland and secreted in high amounts by 60% of pheochromocytomas (16)(17)(18)(19). Nevertheless, we found that NPY is a poor marker for pheochromocytoma because the sensitivity of NPY for detecting the tumor is only 41% (20).
We took the opportunity of surgical removal of seven pheochromocytoma tumors to study the kinetics of elimination of serum metanephrines, catecholamines, and NPY. Our goals were to establish the half-life of circulating NPY and also to assess the elimination rate from blood of catecholamines and metanephrines.
Materials and Methods
patients
We studied seven patients (three women and four men) suffering from hypertension associated with one or several clinical signs suggesting a pheochromocytoma. The urinary collections for free catecholamines and total metanephrines showed increased secretions for at least one of these analytes in all patients, with NE and E concentrations ranging between 1481 and 4700 nmol/day (NE upper reference limit, 620 nmol/day) and between 409 and 1703 nmol/day, respectively (E upper reference limit, 110 nmol/day), and NMN and MN concentrations ranging between 3.5 and 21 μmol/day (NMN upper reference limit, 2.5 μmol/day) and between 1.3 and 8 μmol/day (MN upper reference limit, 1 μmol/day), respectively. In patient 1, NE urinary excretion was within the reference interval (450 nmol/day), and patient 4 had preoperative E and MN urinary excretion rates (42 nmol/day and 0.25 μmol/day, respectively) within the appropriate reference intervals. No urinary collections were available for patient 2.
Abdominal scans showed an adrenal mass in all patients with typical uptake of [131I]-metaiodobenzylguanidine. The histologic analysis of the removed tumor confirmed the diagnosis of pheochromocytoma.
All patients were premedicated before surgery with a combination of α- and β-adrenergic blockers. Surgical removal of pheochromocytoma was carried out under combined anesthesia with fentanyl and a myorelaxant agent.
sampling
A percutaneous venous cannula (Venflon; Becton Dickinson) was inserted into the radial vein for blood sampling for NPY, catecholamines, and metanephrines. The blood samples were drawn at the induction of anesthesia and after the incision. The other samples were usually obtained after the clamping of the blood supply to the tumor by the surgeon (t0) and 1, 2, 5, 7, 10, 20, 30, 40, 60, 90, and 120 min after clamping. When possible, late samples were drawn at 24 h after the end of the operation. The blood was collected in ice-cold 10-mL EDTA tubes (Vacutainer; Becton Dickinson) and centrifuged immediately at 2000g for 20 min at 4 °C. Plasma was separated, and 50 μL of 5 mmol/L sodium metabisulfite was added to prevent catecholamine oxidation. Plasma was stored at −80 °C before all assays, which were usually carried out within 1 month.
amplified enzyme immunoassay of NPY
The amplified enzyme immunoassay of NPY was performed as follows (21): The microplates (Polysorp; Nunc) were coated with 100 μL/well of the monoclonal antibody NPY02 (50 ng) diluted in 50 mmol/L Tris (pH 7.5) for 16 h at 4 °C. Plates were washed three times with Tris buffer containing 0.8 g/L Tween 20 (buffer A) and incubated for 30 min with 200 μL of buffer A containing 50 g/L nonfat dry milk. After four washes with buffer A, wells were filled with 100 μL of plasma sample. The calibration curve was constructed with a plasma pool depleted of endogenous NPY by affinity chromatography with NPY04, an anti-NPY monoclonal antibody not involved in the ELISA.
After a 16-h incubation at room temperature, the plates were washed four times with Pan-Wash buffer (Diagnostic Biosystems). The alkaline phosphatase conjugate of NPY05 (100 μL; 4 ng of antibody) diluted with Tris buffer containing 0.8 g/L Tween 20 and 50 g/L nonfat dry milk was then added to each well, and the plates were incubated for 7 h at room temperature. The plates were then washed twice with Tris buffer containing 2.5 g/L Tween 20 followed by two washes with Tris buffer containing 150 mmol/L NaCl. Bound alkaline phosphatase was revealed by the addition of 50 μL of the substrate. The amplifier (50 μL) was added 45 min later, according to the “Immunoselect kit” recommendations. The absorbance at 492 nm was measured kinetically by a Molecular Devices microplate reader (Molecular Devices), and the data were analyzed by a computer (Soft Max; Molecular Devices). In 303 healthy subjects, plasma NPY concentrations were <0.5 pmol/L in 67% (n = 203), 0.5–5 pmol/L in 25% (n = 76), and 5.1–30 pmol/L in the remaining 8% (n = 24). The mean (SD) plasma NPY concentrations in the healthy subjects was 2.4 ± 2.7 pmol/L. A value of 8 pmol/L (95th percentile value of the healthy subjects) was considered as the upper limit of the reference interval (19).
catecholamine determination
Free catecholamines were determined by liquid chromatography with amperometric detection. One milliliter of serum or calibrator, with dihydroxybenzylamine (Sigma) as internal standard, was extracted on activated alumina at pH 8.6. The alumina was allowed to settle, the supernatant was aspirated, and the alumina was washed three times with water. The catecholamines were then eluted with 170 μL of a mixture of 0.2 mol/L acetic acid and 0.04 mol/L phosphoric acid (8:2 by volume), and 30 μL was injected into the chromatography system. The catecholamines were then separated by chromatography on a reversed-phase column (Nucleosil C-18; 25 cm × 4.6 mm; 5-μm bead size; Macherey-Nagel AG), with a mobile phase of 0.1 mol/L phosphate buffer containing 0.3 mmol/L EDTA, 1 mmol/L sodium octyl sulfate (as an ion-pairing agent), and 30 mL/L acetonitrile (pH 3.5) at a flow rate of 1 mL/min. The electrochemical detector (Model 460; Waters) was set at +0.8 volt. The following order of elution was observed: NE, E, dihydroxybenzylamine, and dopamine. The recovery was 70%, and the detection limit was 10 pg/injection. The interassay CVs were 11% for NE (4.2 ± 0.5 nmol/L) and 12% for E (2.5 ± 0.3 nmol/L). The reference values in normotensive subjects are <4 nmol/L for NE and <0.5 nmol/L for E, respectively.
determination of plasma total metanephrines
Metanephrines were measured as described previously (9). One milliliter of serum containing 50 μL of the internal standard 3-methoxy-4-hydroxybenzylamine was treated with 200 μL of 2 mol/L perchloric acid, vortex-mixed for 10 min on ice, and centrifuged for 10 min at 1800g. The supernatant was further hydrolyzed for 10 min in boiling water. Metanephrines were then adsorbed on a cation-exchange column (Bio-Rad) and eluted with 2 mol/L ammonia containing 200 mL/L methanol. The eluate was evaporated to dryness, and the residue was resuspended in 150 μL of 1 mol/L acetic acid. Metanephrines were separated by HPLC on a reversed-phase column (Nucleosil C-18; 25 cm × 4.6 mm; 5-μm bead size; Macherey-Nagel), with a mobile phase containing 160 mmol/L monochloroacetic acid, 2 mmol/L EDTA, 0.24 mmol/L sodium octyl sulfate (an ion-pairing agent), and 33 mL/L acetonitrile (pH 2.8) at a flow rate of 1 mL/min. The electrochemical detector (Model 460; Waters) was set at 0.78 volt. The recovery for metanephrines was 80%, and the detection limit was 40 pg/injection. The interassay CVs were 13% for NMN (24.5 ± 13.5 nmol/L) and 11% for MN (24 ± 11.7 nmol/L). We had previously determined the mean value for total plasma NMN and MN in a reference group of 18 healthy control subjects, 33 patients suffering from hypertension, and 11 patients with terminal renal failure on hemodialysis (9). The mean (± SD) plasma concentration distributions of NMN and MN for the control group without hypertension were 6.18 ± 2.74 nmol/L (range, 0.1–10 nmol/L) and 2.1 ± 1.52 nmol/L (range, 0.5–5 nmol/L), respectively. In the hypertensive group, the mean values for NMN and MN were 13.8 nmol/L (range, 5–38 nmol/L) and 6.8 nmol/L (range, 0.1–17 nmol/L), respectively. Because mean plasma metanephrine concentrations were higher in patients with abnormal renal function [NMN and MN concentrations, 301 nmol/L (range, 75–558 nmol/L) and 170 nmol/L (range, 77–362 nmol/L), respectively], patients having high creatinine concentrations were excluded from the present study (creatinine concentrations, 69–89 μmol/L; reference values, 44–106 μmol/L). Thus, upper reference limits were calculated from the mean ± 2 SD of the data. The upper reference limits for NMN and MN were 11.6 and 5 nmol/L, respectively (9).
data analysis
A positive result for the measurement of plasma NMN, MN, NE, E, or NPY in a patient with pheochromocytoma was defined as a value equal to or higher than the respective upper reference limit.
The results of catecholamine, metanephrine, and NPY determination in each patient were graphed vs the time. The area under the concentration–time curve (AUC) was calculated using the trapezoidal rule, from the time of tumor vessel clamping up to 120 min. When no sample was available at 120 min, the AUC was estimated by linear interpolation. This AUC value was divided by 120 min and expressed as a percentage of the upper limit of the reference interval, thus providing an average relative degree of increase of the marker over the 2 h following tumor excision. The area under the first moment of the curve (AUMC) was also calculated. The ratio of AUMC over AUC was used as a measure of the “persistence” of increased concentrations of the marker. [In the pharmacokinetic literature, this calculation gives the “mean residence time” of a drug (22); endogenous production, however, precludes a simplistic interpretation of this approach in the present situation.] The product of increase and persistence was chosen as a criterion for the “global performance” of each marker. Indeed, the clinical usefulness of a tumoral marker is related to the height of its increase over the reference interval and to the time it takes to decrease after tumor resection. The increase, persistence, and global performance of the five tumoral markers were compared by means of a Friedman nonparametric analysis of variance, followed by comparisons between the markers with the Wilcoxon signed-rank test. Correlations between the different markers among the samples were evaluated by Spearman rank correlations.
Only the values of NPY were amenable to curve-fitting by a monoexponential decay model, starting from a preclamp plateau and reaching a residual flat concentration. A half-life estimate was derived in each patient from the log-linear slope term of the model.
Results
catecholamine, metanephrine, and NPY concentrations at resection time
Plasma E concentrations were within the reference interval in two patients (patients 2 and 4), whereas they were increased by 5- to 30-fold in the remaining patients. NE concentrations were increased by 2- to 12-fold in all samples at time of resection (t0); however, patients 1, 2, and 3 had values in the reference interval in anterior samples (Fig. 1⇓ ).
Individual profiles for E, NE, MN, and NMN in seven pheochromocytoma patients after the afferent vessels were clamped and the tumor was resected.
Each symbol represents a patient: ▪, patient 1; ▴, patient 2; ▾, patient 3, •, patient 4; □, patient 5; ▵, patient 6; ▿, patient 7. The dashed line represents the upper reference limit.
Plasma concentrations of NMN were increased 5- to 40-fold at time of resection, taking into account that the upper limit of our reference values for NMN was 11.6 nmol/L. Plasma concentrations of MN were increased 3- to 80-fold in six patients, but patient 4 did not exhibit increased concentrations of E and MN (Fig. 1⇑ ).
Plasma NPY concentrations were all above the upper reference limit by 2- to 465-fold (Fig. 2⇓ ).
Individual NPY profiles in seven pheochromocytoma patients after the afferent vessels were clamped and the tumor was resected.
Each symbol represents a patient: ▪, patient 1; ▴, patient 2; ▾, patient 3; •, patient 4; □, patient 5; ▵, patient 6; ▿, patient 7. The dashed line represents the upper reference limit.
catecholamine, metanephrine, and NPY concentrations after removal of the tumor
Catecholamine concentrations.
As depicted in Fig. 1⇑ , E concentrations dropped only in patients 5 and 7 after resection of their pheochromocytomas. These patients exhibited the highest E concentrations at clamping time. Concentrations of E remained increased in patients 1, 3, 4, and 5 for at least 2 h, but returned to below the upper reference limit after 6 min in patient 7.
In contrast to our observation with E, NE concentrations decreased in a time-dependent manner in the plasma of all patients 20 min after resection of the tumor (Fig. 1⇑ ) and then fluctuated between the upper reference limit and values twofold above this limit.
Metanephrine concentrations.
As shown in Fig. 1⇑ , metanephrine concentrations remained increased in all patients but patients 2 and 4 for MN, even 2 h after resection of the tumor. In patient 4, NMN had returned to concentrations within the reference interval 24 h later, whereas this increase persisted 24 h after tumor removal in patient 5 and 48 h after tumor removal in patients 1 and 3. A sample taken from patient 6 six days after resection still revealed MN and NMN concentrations of 4 and 18 nmol/L, respectively.
NPY concentrations.
Plasma NPY concentrations returned to the reference interval within 60–200 min after removal of the tumor (Fig. 2⇑ ), depending on the concentration of the peptide at the time of resection.
correlations between the markers
Two-by-two relationships between the various markers measured in all the study samples are shown in Table 1⇓ . Although several correlation values reached statistical significance, the strength of the associations remained modest. Moreover, the parent markers (E and MN or NE and NMN) did not appear significantly intercorrelated.
Correlation between the concentrations of E, NE, MN, NMN, and NPY in 69 samples provided by seven pheochromocytoma patients undergoing tumor resection.
kinetics of catecholamines, metanephrines, and NPY after tumor removal
The various tumoral markers were compared regarding their evolution during the 120 min after tumor exclusion, using a nonparametric approach. The markers differed significantly about their average relative increase over the upper limit of the reference interval (P = 0.05, Friedman test), with NE and E having the lowest scores and MN and NMN the highest scores (Table 2⇓ ). Two-by-two comparisons indicated significant differences (P <0.05, Wilcoxon test) between the catecholamines and the metanephrines, whereas NPY was intermediate and did not depart from any other marker. The persistence of increased values, reflected in the ratio of AUMC over AUC, also differed significantly among the markers (P = 0.002), with NPY having the lowest score (P <0.02 vs all other markers) and NE appearing significantly lower than NMN (P = 0.008). The global performance (product of increase × persistence) differed significantly among the markers (P = 0.05), with higher values (P <0.05) for the metanephrines compared with the catecholamines. The metanephrines also tended to perform better than NPY.
Summary variables reflecting the evolution of E, NE, MN, NMN, and NPY in seven pheochromocytoma patients after tumor exclusion.
Fitting the data with a monoexponential decay model was possible only for NPY because the values decreased monotonically after tumor exclusion, following a discernible log-linear pattern (see Fig. 2⇑ ). The mean value for the half-life of NPY was 12.3 min (SD, 7.8 min; median, 10.4 min), with individual estimates ranging from 4.7 min (patient 6) to 25.4 min (patient 3).
Discussion
In this study, we confirmed the high sensitivity of plasma total MN and NMN (86% and 100%, respectively) for the diagnosis of pheochromocytoma, in agreement with our previous findings in 10 patients (9) and those of Lenders et al. (10) in 52 patients with sporadic pheochromocytoma, where no patients had plasma concentrations of both free MN and NMN within the reference intervals. Before surgery, plasma and urinary MN were increased in the same patients, indicating a very close diagnostic value for both markers. Therefore, the benefit of measuring plasma MN is related to the fact that there is no need for urine collection or for acidification with hydrochloric acid, with its accompanying problems (23). Interestingly, NE and E concentrations before clamping of the tumor vessel ranged from normal or slightly increased to a fivefold increase in two patients, reflecting the high degree of fluctuation linked to catecholamine secretion. In contrast, the slope of NMN elimination appeared to be relatively flat at all sampling points prior to or even several hours after resection of the tumor, as reflected by both the increase and the persistence of this marker compared with NE. We were unable to establish a similar pattern for MN because sympathoadrenal activation attributable to stress or medications after removal of the pheochromocytoma induced a nonspecific (not related to the tumor) increase in E secretion. Consequently, it was also more difficult to provide an estimated half-life for E.
One limitation of our study is that the nontumoral origin of catecholamines and metanephrines may interfere with our model; the fact that plasma NE concentrations decreased sharply after removal of the tumor and then rebounded to a value higher than the upper reference limit might be explained by the fact that 7% of plasma NE is derived from the adrenal glands and 93% is derived from sympathetic nerves (14). Indeed, we observed in these patients a plateau in plasma NE concentrations (slightly above the upper reference limit) occurring 30–90 min after the removal of the tumor. This conceivably reflects a sympathetic stimulation such as a stress-induced increase of plasma catecholamines that may have occurred upon awakening. Nevertheless, this increase marginally affects the conclusions of this study because the kinetics of catecholamines have been estimated by the AUC determination up to 120 min after clamping of the tumor.
There are several reasons that plasma catecholamines are less sensitive than plasma metanephrines for the diagnosis of pheochromocytoma. (a) Evidence suggests that catecholamine secretion is different from that observed for metanephrines (12). Our data are in agreement with these conclusions based on the fact that high plasma concentrations of metanephrines were observed in our patients with pheochromocytoma who had plasma concentrations of catecholamines within the reference interval in the same sample. For example, patient 2 had high MN concentrations but E concentrations within the reference interval despite tumor manipulation. (b) Previous reports have shown that intravenous infusion of catecholamines produces increases in plasma concentrations of metanephrines that are <6% of those of the precursor amines (13). Thus, peripheral metabolic conversion of catecholamines to metanephrines is unlikely to be responsible for high plasma metanephrine concentrations in patients with a pheochromocytoma. (c) Adrenal glands, like pheochromocytoma, express catechol-O-methyltransferase and are a major site of catecholamine conversion in metanephrines (12)(13). Nevertheless, local transformation of catecholamines to MN within the tumors may provide one, but not the only reason for the superior sensitivity of measurements of plasma NMN and MN compared with those of plasma and urinary catecholamines. The longer plasma half-lives of sulfate-conjugated metanephrines than free catecholamines are consistent with the nature of sulfate-conjugated metanephrines as end-products of catecholamine metabolism, the circulatory clearance of which is dependent on elimination by the kidneys. This, however, contrasts completely with the circulatory clearance of free NMN and MN. The clearance of these metabolites is largely dependent on extraneuronal uptake, the same mechanism responsible for decreases in circulating catecholamines (14)(24). Moreover, it has been reported that free metanephrines have rapid circulatory clearances that are close to those observed for catecholamines (14). Alternatively, MN might be released from unknown compartments (25). This finding indicates that measurements of metanephrines are more sensitive for the diagnosis of a pheochromocytoma than those of plasma catecholamines. However, not all catecholamine-secreting tumors are pheochromocytomas of adrenal origin, and these conclusions may not apply to dopamine-secreting neuroblastomas.
All patients exhibited high plasma NPY concentrations before surgery. This result is in disagreement with a previous report from our laboratory showing that only 60% of pheochromocytomas secreted NPY (19). Nevertheless, the NPY plasma concentrations observed in patients 1 and 3 were increased only two- to three-fold over the upper limit reference for this marker at resection time, but were both found to be within the reference interval in several samples before anesthesia for patient 1 and just above the reference limit for patient 3 (Fig. 2⇑ ). NPY is stored in sympathetic nerves and is known to be released, like catecholamines, from nerve endings during stress (15); therefore, the increase in plasma NPY found in these two patients might originate from nerves rather than from the tumor. NPY was the only marker that allowed fitting for half-life estimation. A half-life of ∼12 min (range, 4.71–16.92 min) was found, which is larger than the 5 min reported by Pernow et al. (26). As described in previous studies, NPY concentrations were highly modified during manipulation of the tumor and returned to reference values within 1 h after removal of the pheochromocytoma (27)(28). During pheochromocytoma surgery, all of the markers measured in this study were only weakly correlated. These results are in agreement with previous studies showing no correlation between NPY and catecholamines (27)(28).
In conclusion, our results indicate that after resection of a pheochromocytoma, total metanephrines are eliminated from the bloodstream in a manner different from that observed for the other analytes measured in this study. Once released from the tumor, free metanephrines might be redistributed and sulfate-conjugated metanephrines stored in an unknown site before elimination. This kinetic phenomenon may contribute to providing high sensitivity and specificity for plasma metanephrines in the diagnosis of pheochromocytoma.
Acknowledgments
The present study was supported by a grants from the Swiss National Fund for Scientific Research FNSRS (Grant 31-53256.98).
Footnotes
1 P <0.01.
1 Upper limit of reference interval.
2 AUC up to 120 min post-exclusion.
3 Area under the moment curve.
4 Ratio of AUC120 min divided by 120 min over NRup.
5 Ratio of AUMC120 min over AUC120 min.
6 Product of increase times persistence.
7 Concentration for NPY, pmol/L.
↵1 Nonstandard abbreviations: NE, norepinephrine; E, epinephrine; NMN, normetanephrine; MN, metanephrine; NPY, neuropeptide Y; AUC, area under the curve; and AUMC, area under the first moment of the curve.
- © 2001 The American Association for Clinical Chemistry
References
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