One key challenge facing studies of the macrolevel context and health is the relative paucity of theoretical development compared with the rapid progress of methodological development [9, 15, 16].

This limitation places epidemiology in sharp contrast with other disciplines, such as sociology, where substantial theoretical work has developed strong frameworks that provide the basis for posing and testing hypotheses on the relations between macrolevel exposures and individual outcomes [17-19].

The principal danger of having underdeveloped theoretical frameworks is that multilevel analyses run the risk of becoming another form of 'black box epidemiology' [20], where the associations between 'exposures' and 'outcomes' are explored, and statistically significant associations are reported, without a theoretical basis for why such associations should exist and why specific factors would cause particular diseases.

Although atheoretical epidemiologic explorations can reveal associations that then generate mechanistic hypotheses that can guide future research [21], they risk unearthing statistically significant relations that represent only the play of chance.

Multilevel analysis in all aspects of health would do well to focus on evaluating specific theoretically driven hypotheses, transcending black box approaches in order to fruitfully identify macrolevel domains that may influence particular health outcomes.

Where no such theoretical or mechanistic work has been conducted for a particular macrolevel determinant, it would be productive for the field if we focused our energy on developing it.


Wight et al. [ 12] present an analysis predicated on a specific neighborhood characteristic (aggregate area-level education) with the theoretical potential to predict a specific outcome (cognitive function).

The authors build their hypothesis on two observations: that educational attainment at the individual level predicts a range of other factors that may be salutary, or deleterious, for health; and that high levels of education are cognitively protective for individuals [22-25].

Therefore, the authors admirably bring together the evidence from previous work, present plausible theoretical reasons as to why the macrolevel determinant of interest may matter for this particular outcome, and use multilevel analysis to assess their hypothesis.

Wight et al. [ 12] also show results from an analysis in which they calculated a composite measure of neighborhood 'disadvantage' and were able to replicate the central neighborhood main effects that are the focus of this paper.

The authors note that they prefer treating neighborhood education as the exposure because the analysis illustrates a specific impact of educational disadvantage.

We suggest that the choice of neighborhood educational attainment as the central independent variable in this analysis is not only preferable but also the only meaningful choice based on the articulated mechanistic pathways, and it is a core strength of this analysis.

However, this discussion also highlights the challenges of discerning the association of one potential macrolevel exposure with an outcome when it is highly collinear with other macrolevel exposures.

There may be causal relations among macrolevel characteristics that need to be better understood before we can untangle their separate and combined influences on health.

Multilevel analyses can move further from atheoretical approaches by explicitly discussing the pathways that may link macrolevel exposures with individual outcomes and, more ambitiously, through attempts at testing these various pathways.

The paper by Wight et al. [ 12] is perhaps more successful than most papers on the first of these counts but, as is often the case in multilevel analyses, is limited on the second.


Wight et al. suggest three plausible mechanisms for explaining the observed association between low neighborhood educational attainment and poor individual cognitive function.

These mechanisms include potential decreased individual activity due to neighborhood stressors, more limited availability of cognitively stimulating material resources such as libraries, and a greater social tolerance for illness.

However, similar to many multilevel analyses, this paper does not explore which of the potential mechanisms might actually explain the observed main-effects association.

In many ways, this shortcoming lays bare an essential tension in epidemiologic analysis more broadly, and one that is particularly germane to multilevel analyses.

The predominant regression modeling techniques are designed to control for potential confounding.

Regression approaches are not designed to consider mediation and are even more ill-suited to differentiating between confounding and mediation.

This issue clearly is relevant to all epidemiologic analysis.

However, it is particularly challenging in the multilevel context, where exposures may be quite distal from the outcomes.

The pressure to demonstrate multilevel associations in models with extensive covariate control is substantial and, to some extent, understandable.

The relative newness of examining distal exposures places an extra onus on investigators to demonstrate that their work is not simply a reflection of unmeasured confounding.

Therefore, it is commonplace in papers that present multilevel analyses to adjust for the individual-level variables that may either confound, but could also plausibly mediate, the observed association between the macrolevel determinant and individual health.

Insofar as macrolevel factors influence individual-level risk factors as well as individual-level outcomes, adjusting for individual-level factors will ultimately render all multilevel associations statistically insignificant [15, 26, 27].

The decision about which variables to include in multilevel models then is a particular challenge faced by analyses interested in explicating the pathways that link macrolevel exposures with individual outcomes.

The analysis by Wight et al. [ 12] is commendable for its judicious use of individual-level controls to reassure us that the findings are not simply a reflection of confounding by well-recognized risks for poor cognitive function without overadjustment for covariates that likely mediate the association of interest.

However, in the interest of testing hypothesized pathways between macrolevel exposures and individual outcomes, methodological development that provides tools to isolate the role of specific mediators of multilevel associations would go a long way toward furthering the contribution of multilevel analyses.


One additional note about this issue is in order.

Suppose that this analysis had included adjustment for limited physical activity, a variable that can plausibly be considered a confounder of the relation of interest but could also plausibly be considered a mediator (as suggested by the authors [12]).

Suppose further that the final model then did not show a statistically significant association between neighborhood education and individual cognitive function.

To what extent would such a model suggest that neighborhood educational attainment is not an 'important' contextual determinant of health?

This question gets at the heart of causal determination and the extent to which, in epidemiology, we are interested in causes distal from the individual.

If a compositional characteristic of a neighborhood—the general level of education—causes less individual activity, and if that lower level of individual activity causes residents in that neighborhood to have lower cognitive function, are we interested only in limited physical activity as a cause, or are we also interested in what might be considered 'causes of causes' [28]? Ultimately, this is not an empiric question as much as a reflection of the values of the epidemiologic research community that influence our analyses and the relevance of our work to the broader public health community.

To some extent, it is self-evident that features of social context influence health.

We epidemiologists must then choose whether we want to contribute to the body of empiric evidence that demonstrates what aspects of social context may matter for what.

We suggest that epidemiology, as the science of public health, might do well to consider causes at multiple levels regardless of their proximity to disease outcomes, providing a broader understanding of the processes that produce health and disease in populations and a wider array of potential points of intervention in the disease process.


Much of our energy in epidemiologic analysis is focused on optimizing our explanatory models, explaining to the extent possible the risk of individual disease as a function of individual 'independent' covariates (exposures), and treating residual variation as random error.

However, as we broaden the epidemiologic lens of inquiry to include determinants at different levels of aggregation, we are beginning to understand that much of the residual variability from individual-level models is not random variability but rather a reflection of a web of interrelations among covariates at multiple levels of influence [10, 29, 30].

Multilevel modeling expands on the individual-level regression approach by including explanatory variables at different levels of aggregation and provides one opportunity to better explore a more comprehensive range of factors that might explain variability in individual health and disease.

Fundamentally, however, multilevel approaches remain concerned with the same goal of optimizing model-based explanation of the likelihood of individual disease.

Regression model-based approaches explicitly endeavor to explain dependent variables (health outcomes) as a function of a fixed set of variables in the hope of providing generalizable explanations.

Relations between covariates at multiple levels of aggregation in nature are unlikely to be unidirectional or simple.

Several authors have called for the broader use of more flexible analytic tools that can take into account mediation, multidirectional causation, and nonlinear relations in epidemiologic research [31-35].

Unfortunately, at this time, most of these methods remain largely inaccessible because of both limitations of most readily available data and challenges in implementing more sophisticated analytic strategies.


Wight et al. [ 12] demonstrate an interaction between neighborhood education and individual education in explaining individual cognitive function.

They surmise that this modification of the neighborhood education–individual cognition effect by individual education might reflect the greater import of social context for those who are already vulnerable, in this case, persons with low levels of education.

The authors suggest that this might guide context-specific health interventions informed by this work.

While this approach is indeed instructive, this demonstration of heterogeneity of association by Wight et al. only scratches the surface of the potential dynamic processes that shape individual cognition in older adults.

Insights from some epidemiologic disciplines such as infectious disease epidemiology [36-39] and, to some extent, the emerging epidemiologic study of frailty among the elderly [40] might lend themselves to broader adoption of epidemiologic methods that account for the complexity of nature across multiple levels.

Broad acceptance of multilevel techniques in the epidemiologic literature presents epidemiologists with a way to consider exposures that extend beyond typical individual exposures or risk behaviors and that may encompass more distal variables that are causes of these more proximal causes of disease.

However, introduction of these methods has also brought challenges for epidemiology.

Methodologically, multilevel analytic techniques force us to consider the analytic difficulties of differentiating between confounders and mediators and of assessing indirect, reciprocal relations as part of a more comprehensive causal framework.

Conceptually, these techniques force us to rethink, yet again, what we might mean by 'cause' and to reconsider the uneasy alliance between the most widely used sufficient-cause frameworks in epidemiology that focus on proximate exposures [41] and the notion of causes of causes.

Through clear exploration of specific associations, and potential heterogeneity within these associations, papers such as the one in this issue of the Journal by Wight et al. [ 12] are a small step in the right direction.

Future epidemiologic theoretical and methodological work will be needed to take bigger steps to satisfactorily address the challenges faced in multilevel analyses.


Galea and Ahern [1] have provided an insightful commentary on our article [2], raising several noteworthy observations pertaining to the current state of and future directions for multilevel epidemiologic research.

We concur that theory-driven multilevel studies are needed, along with explication of the mechanisms that link levels of influence on health, particularly since these mechanisms may be more readily amenable to intervention than structural determinants of health.

For example, in our analyses of the association between neighborhood education and late-life cognition [2], we recommend promoting educational attainment in the general population in disadvantaged neighborhoods because it may prove cognitively beneficial to aging residents of these neighborhoods.

An argument could be made instead for promoting alteration to the hypothesized mechanisms by which we believe educational attainment would benefit aging residents, such as engaging in physical and social activities or increasing access to cognitively stimulating material resources, because these mechanisms are less wieldy from a policy standpoint and are more proximal to the target population.

However, without longitudinal data to test the impact of these mechanisms, this possibility is purely speculative.

By specifying causal mechanisms, theory-based investigations are well poised to distinguish mediators from confounders or analytic noise.


As Galea and Ahern [1] note, a drawback to implementing theory-based multilevel investigations concerns the current lack of appropriate data.

With growing evidence for apparent area-level effects, there will hopefully be greater impetus to develop data sets that specify the potential mechanisms linking levels of influence and that account for the inherent nesting of individuals within contexts.

To date, many multilevel studies have been undertaken with data not originally designed to assess multiple levels of causality (as was the case with data from the Study of Assets and Health Dynamics Among the Oldest Old (AHEAD) analyzed in our study [2]), thereby hindering examinations of heterogeneous associations or limiting the set of variables available for inclusion in multivariate models.

For example, the power to detect cross-level interactions is limited when there are too few cases per macro-level unit to provide sufficient variability in variables of interest.

Although there are approaches for solving the problem of "data sparseness" (e.g., cluster analysis, sample stratification), the problem would best be addressed by designing projects, a priori, to contain ample data points per contextual unit.

Similarly, testing causal mechanisms between levels can occur only if these pathways are operationalized as variables.

Analysis of data sets specifically developed to test such pathways would represent a sizable improvement over secondary analysis of data sets developed for other purposes, which leave the investigator with the option of making use of only those constructs that happen to be available.


By deriving theory-driven research models and applying them to appropriate data sets, future multilevel epidemiologic studies stand to make substantial contributions to our understanding of not only why but how contexts matter to health.


In 1977, Professors Paul Horowicz and Paul De Weer (my Washington University colleague), the founding editor and associate editor, respectively, of AJP Cell Physiology, invited me to write a review article for their new journal.

Paul De Weer, an Na ϩ pump expert, and I had discussed my ideas about the role of Na ϩ pumps and a postulated Na ϩ pump inhibitor/ natriuretic hormone in the pathogenesis of hypertension.

The present article is a personal account of the origin of those ideas and the developments in the field since my hypothesis was published in the journal's first volume (29); Table 1 provides a timeline.


Two key early influences were my experiences treating heart failure (HF) patients with cardiotonic steroids (CTSs, e.g., digoxin) as a medical student and intern, and my 18 months in Daniel Tosteson's laboratory studying the Na ϩ pump, where I routinely used inhibition by the CTS ouabain (pronounced, "wä=ba n") (255) to measure pump activity.

In 1958 I first met Tosteson who told me that "the Na-K-ATPase (NKA, or Na ϩ pump) has just been discovered (264).

You'd better hurry up and work on it before everything about the pump is found out and there's nothing left to do"--so I went to work in his laboratory on a stipend from an NIH training grant.

I'm still working on the pump 60 years later (43)!


My research and clinical experience with ouabain led me to ponder the question of how a pump inhibitor could enhance cardiac contraction (the positive inotropic effect) and thereby benefit patients with HF.

The story of how I fortuitously found the answer by discovering the Na/Ca exchanger (NCX) (14), the "missing link," while studying squid axon Na ϩ pumps as an NIH Special Fellow in Cambridge and Plymouth, England, is described elsewhere (28, 35).

When we discovered the NCX, our 1973 paper (245) we postulated a "privileged communication" between the extracellular fluid and the sarcoplasmic reticulum (SR), involving the PM NCX (and, presumably, PM Na ϩ pumps and the tiny intervening space between the PM and SR).

A similar idea, the ASM "superficial buffer-barrier", was later proposed by Van Breemen (285).

These views were bolstered by evidence for local Na ϩ gradients in heart (298) and ASM (235) and by discoveries about the subcellular localization of Na ϩ pumps and NCX in PM microdomains (see The Plot Thickens-A Lot, below).

This concept differs from the more widely distributed "fuzzy space" (165) that has recently been questioned (90, 187).


By the 1960s the role of dietary salt in the pathogenesis of essential hypertension (49) and its treatment with natriuretic agents (83) were well established, but the underlying mecha- nisms were unknown.

In the late '60s and early '70s Guyton and his coworkers focused on the key role of the kidneys.

They proposed that salt retention and plasma volume expansion led via arterial "autoregulation" to increased vasoconstriction and total peripheral vascular resistance (TPR) and thus to elevated BP (47, 108I jumped into this abyss in 1977 with my AJP Cell Physi- ology article (29).

One of its core concepts was the idea that the functional-coupling of Na ϩ pumps and NCX provides a unique connection between Na ϩ metabolism and ASM cell Ca 2ϩ 1 Glossary of abbreviations appears at the end of the article.


␣2 NKA ouabain binding sites modulate basal BP* Table 3 regulation and, thus, a link to arterial contraction, TPR, and BP.


While scouring the literature for relevant clinical data, I found a number of reports that raised the possibility that a natriuretic hormone, or "third factor," might be involved (37, 45, 54).

Some studies even suggested that this factor was an "active sodium transport" (i.e., Na ϩ pump) inhibitor (72, 80) [the senior author, Neal Bricker, had taught me renal patho- physiology and clinical nephrology when I was a medical student!].


I leaped at the idea and wove a seemingly logical (but rather naïve) story about how salt retention, by an un- known mechanism, triggers the secretion of an endogenous Na ϩ pump inhibitor (i.e., a ouabain-like compound or "OLC").

I was unaware that Szent-Gyorgi had postulated an endogenous CTS 24 years earlier (274), and that Dahl had already shown that a "humoral agent" was instrumental in "salt" hypertension in rats (50).


The OLC would be expected to: 1) inhibit Na ϩ pumps in the kidneys, thereby promoting natriuresis to com- pensate for the salt retention; and 2) inhibit Na ϩ pumps in arterial myocytes, thereby promoting Ca 2ϩ entry via NCX [see Fig. 1, taken from the original article (29)].


The myocyte Ca 2ϩ retention should, of course, increase arterial tone, TPR and BP.


In fact Mason and Braunwald had shown that local ouabain infusion increases forearm vascular resistance in humans (198).


Numerous novel findings have demonstrated that the model in Fig. 1 is overly simplistic.


Nevertheless, the concepts of functional Na ϩ pump-NCX coupling and an OLC with vasotonic (and cardiotonic) actions have now been verified, and many of the details have been filled in.


My 1977 paper (29) was published while I was abroad.

Upon my return, my laboratory colleagues (at Washington University Medical School in St. Louis, MO) all stopped what they were doing to watch me enter my office at the back of the laboratory.

They wanted to see my astonishment when I opened the door and found my desk buried under a mound of several thousand reprint request cards (the article has been cited Ͼ1,700 times).

Interest in those ideas was soon reflected in de Wardener and MacGregor's review, "Dahl's hypothesis that a saluretic substance (plays a) role in essential hyperten- sion" (55), and in Kaplan's reference to my "biochemical hypothesis" (147).


While my AJP Cell article was in press, Haddy and Over- beck (113) also proposed that an OLC is a critical player in the pathogenesis of salt-sensitive hypertension.

They, however, suggested that the critical effects of pump inhibition were reduced electrogenic Na ϩ pump current and consequent myo- cyte depolarization (112), but this mechanism cannot work in the steady state.

Partial pump block will cause a transient decline in Na ϩ transport (and pump current), but when [Na ϩ ] CYT rises, pumped Na ϩ efflux must recover and must equal Na ϩ influx in the steady state to avoid continued net gain of Na ϩ (24).

In other words, chronically, partially inhibited pumps operate with a higher [Na ϩ ] CYT but unchanged pump current.


These proposals about the role of an OLC in hypertension spurred interest in purifying and identifying this compound.

One consequence was that a postdoctoral applicant, John Hamlyn, arrived at my office in 1981, shortly after I moved to the University of Maryland School of Medicine in Baltimore. He told me he wanted to purify this new hormone. I thought to myself, "Sure, you and who else?" Nevertheless, he had some good ideas so I agreed to take him on and let him try, perhaps swayed a bit by the fact that he grew up in the '60s in Plymouth, UK, the site of our NCX discovery.

Little did I- or any of us-realize how terribly difficult this quest for the holy spirit would be.


Most opportunely, shortly after John's arrival I was intro- duced to Avinoam Kowarski and Bruce Hamilton, two clinical endocrinology colleagues who were studying hypertension and had collected plasmas from a number of patients and controls.

They agreed to collaborate and provided us with de-identified plasmas, and John devised a direct but complex biochemical assay to measure Na ϩ -K ϩ -ATPase (NKA) inhibitory activity in the plasmas.

On several occasions I hovered over John's shoulder as he performed the assay, and I marveled at his skill; it left me with no doubt about the quality of his data.

My mentors, Howard Schneiderman, Dan Tosteson, David Gold- man and Alan Hodgkin (26) had imbued me with the need to


When all the results had been collected, John and I sat in my office and plotted the BP as a function of NKA inhibitory activity.

The results were almost too good to believe: although the signals were generally small and there was a fair amount of scatter, there appeared to be a virtually linear correlation between BP and NKA inhibitory activity (Fig. 2)!

The paper was published in Nature (123), accompanied by a "News and Views" commentary (96).

Our article whetted everyone's ap- petite and intensified the search for the endogenous OLC.

In fact, three major pharmaceutical companies contacted me about the possibility of a collaboration.

Scientists at the Upjohn Company (Kalamazoo, MI), led by Jerry Zinn and Donald DuCharme, proposed a joint effort, and I decided to work with them (an excellent choice in retrospect, as our Upjohn col- leagues were wonderful people, and excellent scientists and colleagues).

The decision to work with Upjohn was cemented when NIH (National Heart, Lung, and Blood Institute, NHLBI) turned down our two grant applications to purify the OLC.

The rationale was (to paraphrase the Summary Statements) "Every- body else is doing this so why do we need another group in the field?" and "Everyone who has tried this has failed, so why waste money on anyone else?"


During the next decade, Upjohn funded and participated in our research as follows: I was tasked with procuring, literally, tons of plasma.

A good source was blood banks at our univer- sity and other nearby large medical institutions that performed plasmapheresis.

John worked out the initial steps of extraction and purification on large, normal phase chromatographic col- umns, but the latter proved unsatisfactory.

Accordingly, sub- sequent work was done on smaller, reverse-phase HPLC (high- pressure liquid chromatography) columns that gave far supe- rior resolution and reliability.

At every step, the eluates were carefully screened for NKA inhibitory activity, and the most promising, high-activity samples were saved for further anal- ysis.

Once false-positive had been excluded and the location of relevant biological activities was understood, the isolation steps worked out in Baltimore were refined and scaled up for batch processing at Upjohn.


In 1987 I attended a meeting in Switzerland and stopped off to visit Reuter in Bern.

Harald asked me if I had seen the latest issue of Biochemical and Biophysical Research Communica- tions.

I hadn't, so he escorted me to the library, retrieved the issue from the rack, and opened it to an article by Tamura and Inagami (278).

I skimmed through the report in which they claimed to have purified an endogenous NKA inhibitor of molecular mass 336 Da, with "striking similarity toѧ ouabain," and was alarmed.

I immediately phoned Hamlyn in Baltimore.

He hadn't seen the article either, so I started to read it to him.

Before I'd gotten very far, he interrupted me: "They missed it!" John, who has an exceptional understanding of steroid chem- istry, recognized that they, too, had seen what appeared to be the same bioactive polar material eluting from their columns, but that they had been led astray by a co-eluting contaminant.

When I returned to Baltimore, I sought John out.

He wasn't in his office, but an 8½ ϫ 11 inch photocopy of an Inagami portrait was hanging from each of his bookcases-and from each of the eye-level shelves in his laboratory.

As John ex- plained, "I want to know who my competition is."


In fact, there was no shortage of competition.

In 1988, a prominent Tokyo group, Goto and Yamada, described a bio- active material in dog plasma that "closely resembles ouabain" (103).

Their extracts, while impure, almost certainly contained OLC, but they, too, were thrown off the scent by a contaminant that had an estimated mass of only 343 Da; ouabain's mass is 585 Da.

The following year, another very active group, Hallaq and Haupert in Boston, described the ouabain-like positive inotropic effects of a partially purified material from bovine hypothalamus (116).

Clearly, the key to winning the "race" was to put sufficient pure material in the hands of an analytical chemist who made a living by identifying small molecules.


By 1989 we had 0.8 g of a very highly enriched OLC with an inhibitory action on NKA indistinguishable from that of plant ouabain (120).

At this point we were close, but not yet there.

The OLC was purified by a factor of~10 billion but wasn't completely pure, and the 0.8 g were insufficient for the best mass spectrometers (MS) at the time.

We needed several micrograms of pure material to be sure not to get caught with a contaminant.

Fred Mandel at Upjohn suggested employing the NKA, itself, as an affinity ligand.

A slurry of NKA was used to bind up the OLC; after washing to remove unbound contaminants, the OLC was eluted from the NKA.

Two more rounds of HPLC were sufficient to obtain pure OLC, Don Ducharme phoned a couple of weeks later to tell me that they had identified the structure: "Are you sitting down? You'd better sit down.

You won't believe this.

It's ouabain!" It took him some time to convince me that he wasn't joking ("Okay, Don, now really, what's the structure?").

I immedi- ately went to John's office to inform him (he was then an assistant professor).

John was just as incredulous; he phoned Don back for confirmation.

The chemistry and mechanism of action had indicated that the endogenous molecule was very "ouabain-like"; nevertheless, we were all stunned that it was "indistinguishable from ouabain" (Fig. 3A) (118).

This manu- script, too, had a rough time.

It was submitted to Nature, and although one referee offered to write a "News and Views" commentary, another had "a gut feeling that (endogenous ouabain, EO) is an artifact." Nature therefore rejected the manuscript so I consulted Professor Joseph Hoffman, a re- spected leader in the Na ϩ pump field, who communicated it to Proceedings of the National Academy of Sciences (PNAS; 118).

Our report merited an editorial, "Welcome to ouabain," in The Lancet (68); the original presentation to the American Heart Association High Blood Pressure Council was reported in The New York Times (7).

John and our Upjohn colleagues also published full details of the EO purification and mass spectroscopy (119, 188, 199).

The PNAS article and the earlier Nature (123) and AJP Cell (29) publications were all included among "The Top 100 Cited Scientific Reportsѧ on Hyperten- sion Research" published in the past century (221).

Regretta- bly, a change in leadership at Upjohn led the company to abandon our project shortly after the discovery of EO.

Our Upjohn collaborators, Don DuCharme, Jim Ludens, Fred Man- del, and Rod Mathews, were dismayed because they described the EO study as "the most exciting project" in their careers at the company.


The skepticism voiced by some colleagues when we an- nounced the result was understandable (a few thought I'd gone off the deep end).

After all, ouabain had been used in Africa as an arrow poison for hunting and warfare since the 3rd century (215).

Why would anyone have expected ouabain to be a normal human hormone?

It seems weird, but doesn't this simply reflect our prejudice because we learned that ouabain is a poison more than a century before discovering that it is a hormone?


Within a decade six groups on three continents had verified by MS and NMR spectroscopy that human, bovine, and rodent materials (plasma, urine, adrenals, and hypothalamic tissue) contain EO (Table 2A); the highest concentrations were found in the adrenals (118).

A few outspoken investigators were unable to detect EO in human plasma and claimed that EO did not exist [Table 2C; (172)].

Regrettably, this distracted many researchers and clinicians outside the immediate field who could not evaluate or, more often, were unaware of the actual data.

In the face of so much positive analytical data, including published MS and NMR spectra of EO (Table 2A), very rigorous experiments are needed to prove its absence.

Could so many fine investigators have found the same artifact in speci- mens from different species?

A more likely explanation is that the authors listed in Table 2C may have not detected EO because they did not replicate the original protocols; e.g., one used homogenized placenta (126) rather than plasma, and the others (12, 59, 173) relied on immunoassays.

In contrast, many of those in Table 2A used the native receptor: i.e., they bioassayed eluates for NKA binding and/or inhibitory activity at each stage of HPLC separation (e.g., refs.

118, 120, 188, 232, 256, 276).


Ouabain, also called "g-strophanthin," is an unusual steroid.

It is highly polar and therefore can be challenging to separate by column chromatography, especially when it is present in a complex matrix such as plasma.

Hamlyn recently identified two ouabain isomers in rodent plasma that are absent from commercial (plant) ouabain, one even more polar than ouabain [Table 2B; (121, 138)].

This is additional evidence that EO is of animal origin.

In fact, one group that was unable to detect EO in human plasma (12) did observe an ion currrent peak in their MS-MS product ion spectra that eluted shortly ahead of ouabain ((289) and see footnote to Table 2 line C4).

That ion current peak contained a product ion with the same mass/ charge ratio as ouabain, and we have therefore suggested that it might correspond to the more polar of two ouabain isomers (27, 117) previously observed in rat plasma by multistage MS (121, 138).


The OLC has also been called a "digitalis-like compound" (CTSs obtain from plants of the genus, Digitalis) or DLC.

As discussed in the next section, however, digitalis-like, very hydrophobic compounds (e.g., digoxin; Fig. 3B) do not mimic the unusually polar ouabain-like CTSs (from flora of the genera Strophanthus and Acokanthera) in some of their key actions.

To date, there is negligible evidence that DLCs are endogenous in mammals.

Plant ouabain comes from the bark of the Somali "ouabaio" tree, Acokanthera ouabaio (alt., A. schimperi), hence the name ("waabaayo" is "arrow poison" in Somali), and from the seeds of the flowering woody vines, Strophanthus gratus and Strophanthus kombé.

The cardiotonic, pulse- slowing effect of juice from S. kombé seeds, which contains ouabain, was discovered by the British botanist, John Kirk, in 1861 (182, 204).

Ouabain was first identified chemically and named by the French chemist, Arnaud, in 1888 (8, 139).


Another related CTS, marinobufagenin (MBG; Fig. 3C), a bufadienolide first identified in amphibia (15), has also been linked to hypertension (73, 74), and elevated levels of MBG and another bufadienolide, telocinobufagin, have been found in patients in renal failure (154).

The linkage to hypertension is based mainly on immunologic (versus analytic) identification and on immuno-neutralization by "Digibind" or "DigiFab," commercial anti-digoxin Fab fragments with much higher affinity for ouabain and digoxin than for MBG (239, 240).

Also, MBG binds preferentially to ␣1 Na ϩ pumps (74, 75, 295).

Here I focus on ␣2 Na ϩ pumps and EO (see The Plot Thickens - a Lot for a discussion of Na ϩ pump ␣-subunit isoforms).

Many of the immuno-neutralization studies men- tioned below indicate that the main endogenous CTS binding site ligand is an OLC.

Nevertheless, there are likely to be other related endogenous Na ϩ pump ligands such as the two ouabain isomers mentioned above (121, 138).


An oft-stated concern about EO is that the biosynthetic pathway is unknown (172), but this is not entirely correct.

The adrenals are an important source of EO because they have high concentrations (118, 256, 277).

Adrenalectomy markedly re- duces plasma EO in rats (118), and patients with adrenal insufficiency have very low circulating EO levels (268).

Also, prolonged immuno-neutralization of EO with custom anti- ouabain antibodies increases adrenal cortical, but not medul- lary, weight by about 40%, suggesting feedback enhancement of EO production (213).


There are several reports of hyperten- sive patients with adrenocortical tumors associated with exces- sive production of EO or endogenous "DLC" (190, 252, 258).

Further, plasma aldosterone and EO are co-elevated in patients with essential hypertension (279).

Also, excision of the adre- nocortical tumors (some may even be "ouabainomas"; e.g., Ref. 190) markedly reduced plasma EO or DLC and attenuated the hypertension (190, 252, 258).

( In this case, as in many other reports, "DLC" is simply used as the generic term for an endogenous substance that binds with high affinity to anti- digoxin fab fragments, rather than a reference to an entity chemically identified as "digitalis-like."]


Primary cultured bovine adrenocortical cells exhibit net synthesis of EO (162, 232), as do the adrenals of awake dogs (36).

Perrin and colleagues (232) showed that the biosynthesis of ouabain, like aldosterone, begins with cholesterol side chain cleavage to form pregnenolone, which is then converted to progesterone.

The more distal steps where the pathway di- verges from that of aldosterone synthesis, including the addi- tion of the unsaturated five-member lactone ring and the hexose, L-rhamnose (ouabain is a rhamnoside; Fig. 3A), still


If EO is the Na ϩ pump endogenous ligand, and if it does promote the elevation of BP, as Haddy and Overbeck (113) and I (29) had postulated, there is a direct test: infuse ouabain into animals to elevate the plasma level and determine if it raises BP.

Accordingly, Hamlyn, Haddy, and their colleagues showed that simply administering ouabain to normal outbred rats for 6 - 8 weeks causes hypertension (314).

This landmark obser- vation has now been replicated in numerous laboratories (re- viewed in Refs.

92, 117).

Because ouabain does not always raise BP in outbred rats, a few investigators have mistakenly concluded that the ouabain-hypertension link is unimportant (92).

This view ignores the evidence that the susceptibility to ouabain-induced hypertension is genetically determined.

As Hamlyn and colleagues showed, by inbreeding the least and the most ouabain-sensitive rats, one can generate a highly ouabain- resistant strain and one that is exquisitely ouabain-sensitive within just three generations (6).


Based on the knowledge that all CTSs are pump inhibitors (255) with a cardiotonic action (291), one might have anticipated that the influence of ouabain on BP depends exclusively on its inhibition of Na ϩ pumps and consequent NCX-mediated eleva- tion of cytosolic Ca 2ϩ [as postulated (29) and see Ref.

In this case, all CTSs, which are also vasotonic (198, 283), would be expected to have a similar hypertensinogenic effect.


When Ham- lyn and his post-doc, Paolo Manunta, infused digoxin and related compounds into rats for 5 weeks, however, the BP did not rise (193, 196), even though these compounds, too, are vasotonic (267, 283).

In fact, when digoxin was infused into ouabain-hypertensive rats, the BP fell; i.e., digoxin behaved like an ouabain antagonist (193, 196).

Despite these novel findings, the original report (193) was initially rejected for publication as a full-length article.

It was only published 7 years later, after another report had verified the antagonistic effect of digoxin on ouabain-induced and salt-depen- dent hypertension (131).

Subsequently, Lichtstein and colleagues corroborated the ouabain-digoxin antagonism.

They showed that in anesthetized guinea pigs given a toxic dose of digoxin, and in isolated cardiac muscle treated with toxic concentrations of digoxin or bufalin, low-dose ouabain attenuated the cardiotoxicity (214).

Also, we later observed ouabain-digoxin antagonism in isolated arteries and in primary cultured neurons (267).

In retro- spect, these results should have been anticipated because digoxin has been used clinically for more than two centuries (301) without any evidence that it is hypertensinogenic.

Some of the clinically beneficial effects of Digitalis steroids might even be explained by (unrecognized) ouabain-digoxin antagonism.


The implications of these straightforward observations are profound!

First, because both digoxin and ouabain similarly inhibit the Na ϩ pump (267, 310), this inhibition alone cannot account for ouabain's hypertensinogenic effect, but an explanation wasn't recognized until a decade later (see The NKA Is More Than a Cation Pump, below).

Second, these data demonstrate that the CTS-Na ϩ pump interactions for digoxin and ouabain are not identical; they support the view that the endogenous ligand is very "ouabain-like" and not "digoxin-like." Third, the results suggest that digoxin or a digoxin-like molecule might be a therapeutically useful antihypertensive.

In fact, digoxin has been used to treat hypertension (1).

( During a luncheon following a seminar I gave at Johns Hopkins University three years ago, a post- doc from India told me that his mother, who had hyperten- sion, had been treated with digoxin. " What happened?" I asked. " Her blood pressure came down" was his reply.) This concept was quickly recognized by others and led Bianchi and colleagues to develop rostafuroxin (PST2238; Fig. 3D), a deriv- ative of digitoxigenin (241) that displaces [ 3 H]ouabain from NKA at concentrations that don't inhibit the ATPase (79).

Rostafuroxin antagonizes ouabain-induced hypertension in rats (79) but may have too low an affinity for the pump (267) to be useful in unselected patients with essential hyperten- sion (269).

This drug, however, effectively lowers BP in hypertensive patients who carry specific isomorphisms (161).

Nevertheless, a more effective EO antagonist is obviously needed.


In a prescient article, Manunta, Hamilton, and Hamlyn (192) identified some of the structural features of ouabain that are required for its hypertensinogenic effect.

They showed that the aglycone ouabagenin is as effective as ouabain in inducing hypertension in the rat.

Surprisingly, modifications of the normally unsaturated lactone ring-i.e., saturation, conforma- tional constraint, or opening of the ring-strengthen the hy- pertensinogenic effect even though these alterations greatly weaken the ATPase inhibitory activity by increasing the EC 50 almost 100-fold.

This, too, indicates that the hypertensinogenic effect is not tightly linked to the Na ϩ pump inhibitory activity but instead suggests that the effect is more closely related to EO-activated signaling mechanisms (see The NKA Is More Than a Cation Pump).


An alternative test of the EO-hypertension hypothesis is to neutralize the circulating EO in hypertensive models (or humans).

Indeed, immuno-neutralization of EO with Digibind significantly lowers BP in rats with DOCA-salt (156) or reduced renal mass (146) hypertension and prevents ACTH-induced BP elevation in mice (60).

By the late 1990s the link between ouabain/EO and hypertension was already well documented in humans (195, 233, 252) and rodents- despite a few awkward and as yet unexplained observations such as why only about half of essential hyperten- sives and patients with aldosterone-producing adenomas have substantially elevated EO levels (252).

Nonetheless, plasma EO correlates directly with BP in essential hypertensives and their offspring (194, 233).


In 1985 Lingrel and Shull cloned the Na ϩ pump catalytic (␣) subunit which contains the transport machinery and ouabain binding site (259).

By the early '90s they had cloned all 4 ␣-subunit isoforms (␣1-␣4) and the small, glycosylated mam- C10 THE PUMP, THE EXCHANGER, AND THE HOLY SPIRIT AJP-Cell Physiol • doi:10.1152/ajpcell.00196.2017 • www.ajpcell.org Downloaded from journals.physiology.org/journal/ajpcell at McGill Univ (132.216.240.244) on May 18, 2024.

malian ␤-subunit isoforms (␤1 and ␤2) (23, 180).


The pumps are ␣␤ dimers, and ␤ is a chaperone that is required for maturation, intracellular routing, and activity of the pump, including ouabain binding (124).

The pump complex may also include a small regulatory "FXYD family" protein, sometimes called the ␥-subunit (91).


Importantly, all cells express pumps with an ␣1-subunit, usually the predominant pumps [80 -90% of total pumps in the heart (19, 299)].

Most cells also express pumps with an ␣2- or ␣3-subunit [the remainder (19, 141, 299)].

In addition to ␣1 pumps, for example, all types of muscle (cardiac, skeletal, and smooth), glial cells, and hip- pocampal neurons also express ␣2 Na ϩ pumps, while most neurons express ␣3 pumps (179, 203).


The ␣1 pumps are "housekeepers" that maintain the low [Na ϩ ] CYT in bulk cytosol and mediate net Na ϩ transport across epithelia (201).


So what do the ␣2 and ␣3 Na ϩ pumps do?


While studying ␣-subunit distribution in ASM cells and neurons, we serendipitously discovered that the pumps with ␣2- or ␣3-subunits were confined to PM microdomains that were closely apposed to "junctional" SR or endoplasmic retic- ulum (S/ER) (144, 145).

The NCX colocalized with ␣2 or ␣3 in these microdomains at PM-S/ER junctions, which we have called "PLasmERosomes" (33).

In contrast, ␣1 pumps were more ubiquitously distributed in the PM (144).

This was the first experimental support for our conjecture that there is preferential communication between the PM and S/ER at the PLasmERosomes (245).

Further, the unusually low affinity of rodent ␣1 for CTSs (218) implicated ␣2 and/or ␣3 pumps and NCX in the cellular action of low-dose CTSs (9, 33).


Crucially, Lingrel's Na ϩ pump-cloning studies opened the door to genetic alteration of the pumps to facilitate the study of ␣ isoform function.

Mutations included the generation of mice with ouabain-resistant (R) ␣2 pumps [␣1 R/R ␣2 R/R mice (48, 61)] and R-␣2 mice with ouabain-sensitive (S) ␣1 pumps (␣1 S/S ␣2 R/R , or "SWAP" mice (63)), to determine the role of the ouabain binding site (179).

The ␣1 R/R ␣2 R/R mice, with two amino acids normally found only in ouabain-resistant rodent ␣1 substituted into the ␣2 ouabain binding site (48), apparently have normal Na ϩ -K ϩ -ATPase activity (61), but they exhibit a baseline cardiovascular phenotype.

Telemetry studies show that baseline mean BP (MBP) is 4 -10 mm Hg higher in awake ␣1 R/R ␣2 R/R than wild-type (WT) ␣1 R/R ␣2 S/S mice (Table 3).

Also, pregnant ␣1 R/R ␣2 R/R dams have about 10 mm Hg lower systolic BP than do pregnant WT controls (228).

Thus, the ␣2 ouabain binding site and, presumably, its endogenous ligand play a role in normal basal BP regulation.


The ␣1 R/R ␣2 R/R mice are resistant to hypertension induced by ouabain, ACTH, elevated intracerebroventricular (icv) NaCl, and high dietary NaCl (60, 62, 168, 185, 286).

Even angiotensin (Ang) II-high salt hypertension, a common hyper- tension model, apparently requires S-␣2 pumps for full expres- sion (25, 187a).

Trans-aortic constriction (TAC)-induced car- diac hypertrophy also is delayed/attenuated in these mice despite the TAC-induced BP elevation (296).

Conversely, SWAP (␣1 S/S ␣2 R/R ) mice, which have 5-10 times as many S-pumps as WT mice, exhibit a smaller BP rise but accelerated TAC-induced hypertrophy and HF (25, 296).

In addition, ACTH-induced and salt-induced hypertension and TAC-in- duced cardiac hypertrophy, but not TAC-induced pressure overload, are all markedly attenuated by immuno-neutraliza- tion of EO (60, 168, 176, 286, 296).

Also, active anti-ouabain immunization with an ouabain conjugate attenuates hyperten- sion in salt-sensitive Dahl rats (101).

Collectively, these sem- inal studies demonstrate that EO and its receptor site partici- pate in the pathogenesis of hypertension, cardiac hypertrophy, and HF.

Moreover, the SWAP mouse data may be relevant to humans and other non-rodent mammals that have a ␣1 S/S ␣2 S/S Na ϩ pump genotype.


I first learned of the ACTH-hypertension results when Jerry Lingrel presented them in a symposium in 2004 honoring Joe Hoffman on his retirement.

Joe, who was sitting in the front row, turned around and gave me a "thumbs-up" when Jerry finished speaking.

It was welcome support for the EO-Na ϩ pump-hypertension hypothesis, when many had anticipated a negative result.


There is a fly in the ointment, however.

The ␣1 R/R ␣2 R/R mice apparently can develop DOCA-salt hypertension despite the ouabain resistance (186).

The discrepancy between this result and the attenuation of DOCA-salt hypertension in rats by immuno-neutralization of EO with Digibind (156) needs to be resolved.

Also, the apparent paradox that ␣1 R/R ␣2 R/R mice have elevated basal BP (Table 3) yet are unable to develop several forms of hypertension requires an explanation.


Several other unexpected and astonishing observations that revolutionized our conception of the CTS-Na ϩ pump endo- crine mechanism were made in the 1990s.

One is the discovery by Xie and Askari and colleagues that ouabain binding to Na ϩ pumps activates protein kinase (PK) cascades and cell signal- ing apparently independent of active cation transport (110, 135, 153, 181, 231, 305).

The two pathways that have been identi- fied, epidermal growth factor receptor (EGFR)/Src/MEK/ ERK1/2 and PI3K1A/PDK/Akt (87, 111, 175, 303), are acti- vated by ouabain in a variety of cell types, including cardio- myocytes, ASM cells, and kidney tubule cells.

Ouabain signaling also generates reactive oxygen species (ROS) (307).

X-ray crys- tallography shows that it lies within the cation transport chan- nel (163, 308).

Another possibility is that Na ϩ pumps function as tetramers of the ␣␤ protomer, i.e. (␣␤) 4 (267).


At first I was quite skeptical that the pump did anything other than pump cations.

After all, I had lived with that classical concept for about 40 years, and it's hard to teach on old dog a new trick (30).

Nonetheless, the discovery that prolonged ouabain, but not digoxin, treatment triggers PK- mediated ion transporter reprogramming, including upregula- tion of NCX1 both in vivo and in vitro in ASM cells (237, 238, 323) and cardiomyocytes (32) was the clincher.

Our initial report was rejected by Journal of Physiology with the claim that EO doesn't exist (yes, it's getting monotonous!), but was published in AJP Heart and Circulatory Physiology (238) with an accompanying "Editorial Focus" (94).

Importantly, here was a possible explanation for the fact that digoxin inhibits NKA and is vasotonic but does not raise BP: The PK-mediated upregulation of NCX1 and enhancement of ouabain-induced, NCX-mediated Ca 2ϩ entry at PLasmERosomes may be crucial for BP elevation (see Ref. 31).

In a striking turnabout, Journal of Physiology recently invited and published our review of the evidence that ␣2 and its ouabain binding site are pivotal in cardiovascular physiology and pathophysiology (31).


Another unanticipated development in the mid-90s was Frans Leenen's discovery of the role of "brain ouabain" in the pathogenesis of hypertension (129, 130, 132).

This followed earlier reports of an OLC in the hypothalamus (275, 282). But why was brain EO necessary, as Leenen's experiments clearly suggested (130), if most of the circulating EO comes from the adrenal cortex (36, 118, 268) where it is synthesized (162), and if circulating EO/ouabain has a direct vasotonic effect (9, 238, 317, 318)? How did brain ouabain fit into the "big picture" if circulating EO directly augments Ca 2ϩ signaling and activates the arterial (and cardiac) PK-dependent Ca 2ϩ transporter re- programming?

Despite these key, unresolved questions, Leenen, and Hamlyn and I, proceeded on our separate ways for more than a decade without trying to reconcile our different viewpoints.

Then, in 2010 Leenen invited me to participate in a satellite conference he organized in conjunction with the International Society on Hypertension's Vancouver meeting.

Following our divergent presentations he asked me if there wasn't some way to reconcile our differing views.


For the first time this got me to think seriously about the dilemma (30).

I invited Frans to Baltimore, and John and I initiated a collab- oration with him.

This led to a jointly authored review in which we postulated that Leenen's novel hypothalamic pathway, involving "brain EO" (88), helps regulate adrenal secretion of EO and circulating EO levels (34).

We then embarked on a collaborative study that tested this hypothesis (121).

Specifi- cally, we found that icv infusion of low-dose Ang II elevated BP and plasma EO and upregulated arterial expression of NCX1 and SERCA type 2 (SERCA2).

All these effects were blocked by icv infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker (121).


Yet despite all this evidence for a novel EO-Na ϩ pump endocrine mech- anism, grant applications, including the renewal of our Pro- gram Project Grant (which generated much of these data) and a new collaborative program with Leenen, were rejected.

For example, despite two enthusiastic reviews of one application, the primary reviewer stated (Summary Statement) that "It is intuitive that these... (central nervous system, CNS) pathways likely existѧ Hence, the innovation is modest at best." Any- thing is obvious when you're given the answer [in this case, the preliminary data that supported a novel explanation for the paradoxical finding that high salt and salt restriction both elevate plasma EO (191)].


In his 1953 monograph, Szent-Gyorgi suggested that in HF there might be a "missing screw" that exogenous CTSs are able to replace (274).

Therefore, following the discovery of EO, Hamlyn, Manunta, and Stephen Gottlieb, a University of Maryland cardiologist, measured EO levels in HF patients.

The results were clear: EO was elevated, not reduced (as implied by Szent-Gyorgi), in HF patients, and the level correlated in- versely with cardiac index (104).

Was this already a hint of EO-digoxin antagonism?

A later corroborative study from Milan demonstrated that EO correlates directly with the extent of cardiac hypertrophy in patients with end stage renal disease (270).

In these patients and in patients undergoing cardiopul- monary bypass surgery (22) the highest EO levels were asso- ciated with the most severe cardiac disease and the worst clinical outcomes (263).


The NIH (NHLBI) funded the Digitalis Investigative Group (DIG) clinical trial (1990 to 1998), a randomized digoxin/ placebo test on 7,788 HF patients, to investigate the effective- ness of digoxin therapy [https://clinicaltrials.gov/ (58)].

The presence of EO and occurrence of ouabain-digoxin antagonism surely raise fundamental questions about the interpretation and significance of this trial's outcome (93, 229).


During the past 15 years it has become apparent that the pathogenesis of hypertension and HF involves renin-Ang- aldosterone system (RAAS)-mediated activation of the same central pathways that drive sympathetic input to the arteries and heart (Fig. 4) (57, 226, 227, 300, 313, 321).

Thus, in retrospect it is not surprising that Leenen found that "brain ouabain" participates in the same CNS mechanisms that en- hance sympathetic nerve activity (SNA) in HF (169) and hypertension (129, 130, 132).

After all, similar agents, includ- ing Ang-converting enzyme (ACE) inhibitors, Ang receptor blockers (ARBs), and MR blockers, are all effective therapies for both hypertension and HF (42, 140, 157, 170).

Further- more, the progression from uncontrolled hypertension to car- diac hypertrophy and then HF is well documented (46, 128, 290, 316).

Additionally, as noted above (The Plot Thickens), the cardiac hypertrophy that results from TAC-induced pres- sure overload in mice also depends in part on the ␣2 Na ϩ pump ouabain binding site and on EO (25, 296).

Indeed, in SWAP mice with S-␣1 Na ϩ pumps the progression to HF is acceler- ated (296).

This is a further manifestation of the EO-ouabain- sensitive Na ϩ pump-hypertension-HF relationship.


Initially, I questioned the EO-HF link, so John Hamlyn and I explored the effects of coronary occlusion in mice.

We found C12 THE PUMP, THE EXCHANGER, AND THE HOLY SPIRIT AJP-Cell Physiol • doi:10.1152/ajpcell.00196.2017 • www.ajpcell.org Downloaded from journals.physiology.org/journal/ajpcell at McGill Univ (132.216.240.244) on May 18, 2024. 32) as others have observed in human and experi- mental HF (219, 271).

Not surprisingly, the signaling mecha- nisms appear to be cell type-specific.


The preceding discussion about the cardiovascular conse- quences of interfering with the EO-Na ϩ pump mechanism raises critical questions about how this system works.

In other endocrine systems, either absence or excess of the hormone can have pathophysiological consequences as in, for example, hypo- and hyperthyroidism.

This is also the case for the EO-Na ϩ pump system; furthermore, the pathophysiological consequences are not always "all-or-none." For example, ouabain resistance in mice delays/attenuates TAC-induced car- diac hypertrophy but does not prevent it (296; see also Ref. 25).

Also, transient or modest elevation of EO may be beneficial (e.g., the cardiotonic effect), but greater and more sustained elevation may have pathological consequences (hypertension, cardiac hypertrophy, and HF).

How can we explain these seeming contradictions?


The key role of the sympathetics in cardiovascular control is widely accepted, but it seems logical that other mechanisms also contribute to these functions that are so vital for survival.

In this regard the EO-Na ϩ pump system operates in conjunc- tion with and in parallel to other mechanisms, especially the sympathetic nervous system (Fig. 4).

Whereas ␣1 R/R ␣2 R/R mice have only a modest baseline BP phenotype (Table 3; see The Plot Thickens), ␣-adrenoceptor blockade markedly re- duces arterial tone (302, 315) and lowers BP (105, 220).

Enhanced sympathetic drive in hypertension and HF is main- tained at least in part by activation of a novel, slow neuro- modulatory pathway in the hypothalamus (Fig. 4, top right corner), one component of which is locally secreted (paracrine) "brain EO" (88, 121, 300).


Interruption of this pathway by lesioning the anteroventral third ventricle (AV3V) could ex- plain why AV3V lesions prevent the maintenance of estab- lished hypertension (39).

The neuromodulatory pathway feeds forward via Ang II onto the brainstem sympathetic system that drives both the heart and arteries (171).

In addition to modu- lating SNA, the slow pathway regulates plasma EO, perhaps via SNA to the adrenals and Ang II (31, 121).

This endocrine mechanism serves as a parallel pathway to the sympathetics in influencing cardiac and vascular signaling (Fig. 4; see also Ref. 31).

Block of the neuromodulatory pathway therefore attenu- ates hypertension and HF by both central and peripheral mechanisms (121, 134) and has obvious implications for hy- pertension and HF therapy (166).


Circulating EO acts directly on the heart and arteries in two ways.

First, by inhibiting ␣2 Na ϩ pumps, EO exerts acute cardiotonic and vasotonic actions [Fig. 4, bottom half); (31)].

Second, by activating PK cascades, EO triggers reprogram- ming of Ca 2ϩ transporter protein expression exemplified by increased NCX1 in both the heart and arteries, and increased arterial but decreased cardiac SERCA2 (see "chronic" path- ways in Fig. 4, bottom).

In arteries with tone, because NCX1 normally mediates net Ca 2ϩ entry, the upregulation of both NCX1 and SERCA2 contributes to enhanced Ca 2ϩ signaling and increased tone and fosters hypertension.

In the heart, however, although NCX1 normally mediates Ca 2ϩ extrusion, primarily, the combination of Na ϩ pump inhibition and in- creased NCX1 expression tends to promote net Ca 2ϩ gain, and thereby increases ventricular "stiffness" and reduces diastolic dilation (Fig. 4; bottom right) (86, 95, 200, 236).

Also, the decreased SERCA2 expression reduces Ca 2ϩ storage and sig- naling (20, 21, 273, 280).

Thus the elevated plasma EO initially contributes to enhanced Ca 2ϩ signaling and cardiac workload, and to hypertrophy by a sustained cardiotonic effect as well as the protein reprogramming.


But, if EO elevation is greatly prolonged, and especially if the EO level continues to rise, this will eventually increase cardiac stiffness, reduce Ca 2ϩ signal- ing, and cause HF (additional details in Ref. 31).

The key role of EO and ␣2 Na ϩ pumps is indicated by the results of studies on TAC-induced cardiac hypertrophy.

Contrary to the dogma that cardiac stretch directly induces the hypertrophy (223, 297, 309), pressure overload-induced hypertrophy is attenuated/ delayed when EO is immuno-neutralized, or its receptor is knocked out or mutated to an ouabain-resistant form (248, 296).


Ouabain-sensitive Na ϩ pumps are expressed in all tissues.

Therefore it is appropriate to ask whether ouabain-resistant mutants and EO immuno-neutralization induce dysfunction in organ systems other than the cardiovascular system.

An affir- mative answer would help validate the EO-Na ϩ pump endo- crine concept.

The CNS might, however, be protected from circulating EO by the blood-brain barrier, which polar EO does not permeate- except at the circumventricular organs, which lack the barrier.

Nevertheless, as discussed next, several groups explored the idea that brain neurons and/or astrocytes might express an EO-dependent phenotype.


Following up on their earlier studies demonstrating learning and behavior alterations in heterozygous mice with a single null mutation in ␣2 (or ␣3) Na ϩ pumps (210), Lingrel and coworkers discovered that ␣1 R/R ␣2 R/R mice have a behavioral phenotype: They exhibit decreased locomotion in a novel environment, a blunted acous- tic startle reflex, enhanced response to methamphetamine (an indirect sympathomimetic), and impaired egocentric learning (254).

Importantly, these specific behavioral and memory def- icits, which are likely related to ␣2 Na ϩ pump expression in hippocampal pyramidal neurons and/or brain astrocytes, differ from those in heterozygous ␣2 mice (␣2 S/Ϫ ) (254).

The ␣2 S/Ϫ mice, which have a null mutation in one ␣2 allele and express only half the normal complement of S-␣2 pumps, exhibit increased anxiety, decreased locomotor activity, and impaired spatial learning (137, 210).

In fact, a difference would be expected because ␣2 S/Ϫ is equivalent to elevation of the EO level (i.e., greater inhibition of ␣2) but reduction of EO- activated signaling, while ␣2 R/R is equivalent to elimination of EO.

Thus the ␣2 ouabain binding site and, presumably, EO are associated with specific brain functions.

Low-dose icv ouabain infusion induces mania-like behavior in rats (69, 70).

Also, immuno-neutralization of EO with icv anti-ouabain antibodies attenuates amphetamine-induced hy- peractivity (another mania model) and is antidepressive in the Flinders Sensitive Line of genetically depressed rats (97, 127).


An important caveat to these experiments, however, is that most neurons express (S) ␣3 Na ϩ pumps which have a differ- ent behavioral phenotype from ␣2.

For example, ␣3 heterozy- gotes (␣3 S/Ϫ ) have spatial learning and memory deficits, and increased locomotor activity and locomotor response to meth- amphetamine (210).

Heterozygous Mishkin mice, with an in- activating point mutation in one ␣3 gene (44), also display mania-like behavior resembling that of bipolar patients in the manic phase, with enhanced exploratory locomotion in a novel environment and increased sensitivity to amphetamine.

These symptoms are treatable with mood-stabilizing drugs such as lithium and valproic acid, and with rostafuroxin (151).

Because ouabain infusion and EO immuno-neutralization should affect both ␣2 and ␣3 pumps, the results of those tests support the view that both EO and its binding sites are important for behavior and learning, but they do not distinguish between the roles of the ␣2 and ␣3 Na ϩ pumps. [ Regrettably, no ␣3 R/R mouse has yet been generated.]


The source of the EO that regulates the neurons involved in behavior and learning is unknown.

Circulating EO might influence hypothalamic neurons in the circumventricular or- gans that participate in cardiovascular control (294), if only as feedback to help regulate the plasma EO level.

However, EO-regulated neuronal activity in other areas of the brain surely require local EO synthesis.

This circumstantial evidence that EO is synthesized and secreted in the brain complements Leenen's findings ("Brain Ouabain"-Really?).


In skeletal muscle, ␣2, not ␣1, Na ϩ pumps are the most prevalent, and the ␣1:␣2 ratio is approximately 1:6 (125), but this can vary depending on fiber type and conditions.

Most skeletal muscle ␣2 pumps are, however, located in intracellular membranes, and exercise training and insulin promote the trafficking of ␣2 pumps to the PM (89, 136, 272).


EO and the ␣2 ouabain binding site modulate exercise endurance.

␣1 R/R ␣2 R/R mice have fewer exercise failures on a treadmill than do WT mice (243).

Conversely, mice with targeted knockout of skeletal muscle ␣2 Na ϩ pumps (SkM- ␣2 Ϫ/Ϫ mice) fatigue faster, i.e., they have more treadmill failures than do WT mice (244).

Further, the increased fatiga- bility of isolated extensor digitorum longus muscle from SkM- ␣2 Ϫ/Ϫ mice is mimicked by treating WT extensor digitorum longus muscle with ouabain (244).

Thus, reduction of EO binding enhances, and inhibition or knockout of ␣2 pumps curtails exercise endurance.


Numerous reports demonstrate that EO is a stress hormone.

Plasma EO is elevated not only in hypertension, and cardiac hypertrophy and failure, but following cardiac surgery (263), in renal failure (122), in ectopic corticotropin syndrome (102), in normal human and rodent pregnancy and even more so in preeclampsia (56, 138), following intense exercise (17), and during salt depletion as well as dietary salt excess (191).

Many reports indicate that low dose ouabain/EO is a neu- roprotective and antiapoptotic agent whose effects appear to be mediated by the Ca 2ϩ -dependent nuclear transcription factor, NF-B (4, 53, 148, 175, 261).

Several in vivo and in vitro studies also show that ouabain/EO has an anti-inflammatory action (53, 160, 224).

On the other hand, some in vivo and in vitro studies indicate that ouabain/EO can activate proinflam- matory responses (148, 150, 152).

For example, in primary cultured glia, knockout of ␣2 Na ϩ pumps by siRNA prevents activation of the NF-kB-mediated inflammatory response to lipopolysaccharides (150).

This is clearly a "hot area", and new studies are needed to elucidate how EO can elicit both pro- and anti-inflammatory responses.

Is this simply a concentration- dependent effect (as seems unlikely) or are other factors involved?


The preceding observations demonstrate that the actions of EO are wide- spread.

Additionally, several groups have shown that low nanomolar ouabain/EO is a growth factor in a variety of tissues (10, 11, 66, 98, 217, 257).

For example, ouabain promotes cyst growth and cystic fluid secretion in renal tissue from patients with an autosomal dominant polycystic disease mutation, ap- parently via the EGFR/Src/MEK/ERK signaling pathway (142, 216).


But is EO a growth factor in vivo? To determine the role of EO in pregnant rats, Lichtstein and colleagues immuno-neu- tralized the EO by intraperitoneal infusion with anti-ouabain antibodies.

This treatment (vs. control IgG) significantly re- tards fetal development and reduces kidney and liver growth (65).

Conversely, prolonged infusion of ouabain in rats in- creases heart and kidney weights, and these effects are abro- gated by rostafuroxin (77).

In vitro, immuno-neutralization of EO in growth medium containing fetal bovine serum attenuates the growth of several types of primary cells in culture (65, 66).

Further, Aperia and coworkers report that malnourishment of pregnant rats by feeding a low-protein diet impairs fetal kidney growth, and that subcutaneous infusion of low-dose ouabain in protein-restricted dams protects fetal development (174).

In vitro, nanomolar ouabain sustains kidney development and attenuates apoptosis of renal epithelial cells and podocytes (40, 174, 175).

These latter effects appear to be due to upregulation of NF-B (174).


It has been suggested that activation of signaling cascades by low-dose (nanomolar) ouabain in rodents is mediated by ␣1 pumps (174, 304), but this is at odds with the finding that ouabain-induced hypertension, which apparently depends on this signaling (31), is averted in ␣1 R/R ␣2 R/R mice (62).

Thus, the low-dose (nanomolar) ouabain/EO-induced effects on ro- dent kidneys needs to be reconciled with reports that R-␣1 is the only Na ϩ pump ␣ isoform detected in this tissue (71, 202).

Others, however, suggest that very low levels of ␣2 and/or ␣3 are also expressed in some rat renal tubule segments (5, 76); also, ␣3 has been observed in rabbit kidneys in which S-␣1 is the predominant isoform (16).

This issue may be settled by the aforementioned report that in mouse glial cells, which express R-␣1 (80 -90% of pumps) and S-␣2 Na ϩ pumps (99), silencing ␣2 with siRNA prevents ouabain from activating PK signaling and the neuroinflammatory pathway (150).

Thus, as in rodent hearts (25, 31) and glial cells, a tiny minority of pumps (the S-␣2 or S-␣3) might mediate the signaling actions of EO in rodent kidneys.

Nevertheless, given these examples of EO acting as a growth factor in the kidneys and liver, and in view of the broad distribution of ouabain-sensitive Na ϩ pumps, it seems that additional examples of this action are likely to be C15 THE PUMP, THE EXCHANGER, AND THE HOLY SPIRIT AJP-Cell Physiol • doi:10.1152/ajpcell.00196.2017 • www.ajpcell.org Downloaded from journals.physiology.org/journal/ajpcell at McGill Univ (132.216.240.244) on May 18, 2024.

identified in other tissues.

Clearly, we have much to learn about this aspect of EO function.


If the ouabain/EO level is too high, however, the hormone can stimulate generation of ROS and trigger apoptosis (66, 158, 284, 306, 307).

Manunta and colleagues found that acute kidney injury, which may occur (e.g.) as a serious complication of cardiopulmonary bypass surgery (250, 251), is associated with high levels of circulating EO (22, 262).

In rats with ouabain-induced hypertension or renal ischemia-reperfusion injury, renal podocyte nephrin, a key component of the glo- merular filtration barrier, and creatinine clearance are reduced and proteinuria is increased.

All these signs of impaired barrier function are prevented or reversed by treating the rats with rostafuroxin (78, 288).


In 1977 I suggested that the endogenous Na ϩ pump inhibitor should be a natriuretic as well as a hypertensinogenic agent (29).

But, is there evidence that the low concentrations in the circulation directly inhibit Na ϩ reabsorption by the kidneys? During intravenous (iv) infusion of anti-ouabain antibodies (passive immunization) in normal rats, a transient anti-natriuresis (i.e., salt retention) was ob- served a day after starting the EO immuno-neutralization while BP remained normal (213).

This suggests that EO may be a natriuretic agent, although the mechanism is unclear because virtually all the Na ϩ pumps responsible for Na ϩ reabsorption in the rodent renal tubule epithelium are R-␣1 (202).

The transience of the effect is expected with constant dietary salt because, following a brief net gain of Na ϩ and attendant anions, a new steady state should soon be achieved so that Na ϩ intake is once again balanced by output.


In an isolated case report, an 11-year-old patient who devel- oped hyponatremia due to salt wasting following the removal of a large brain tumor was treated with iv Digibind; this reduced the natriuresis and corrected the hyponatremia (205).

Here, too, the mechanism for the anti-natriuresis was not resolved but, in this case, could have been due to immuno- neutralization of nanomolar EO that normally binds to ␣1 Na ϩ pumps in the human kidney tubule epithelium.

Furthermore, saline loading enhances natriuresis in SWAP mice, and the fact that this effect is attenuated by infusion of Digibind (184), indicates that EO is natriuretic.


In sum, the data from mutant mice and from EO immuno- neutralization studies are powerful evidence that EO, and ␣2 and ␣3 Na ϩ pumps, are components of a major endocrine/ paracrine system with critical cardiovascular, renal, and CNS influences (31, 179).

This endocrine system has been largely overlooked, perhaps in part because of the insistence by some that "endogenous ouabain is not ouabain" (Ref. 172; see Confirmation-Despite a Few Vocal Naysayers).

In practice, however, the exact structure of EO seems secondary to vali- dating the concept of an EO-Na ϩ pump endocrine system.


Circulating EO, like most hormones, is expected to have a normal physiological concentration range.

Plasma levels that are too high or too low are pathophysiological as summarized in this review: e.g., elevated EO leads to reduced cardiac index (104) and increased left ventricular hypertrophy (234), and low EO impairs fetal development (65, 174).

In the latter case, ouabain would appear to be the ideal replacement therapy [recall Szent-Gyorgi's "missing screw" (274)] provided that the normal plasma level and the appropriate dosage are known.


While they are equipotent (64, 267, 311, 312), ouabain acts much more rapidly than digoxin (81) and the absorption (only Ϸ2-2.5% vs. Ϸ70 - 80% for digoxin) and bioavailability of orally administered ouabain (106) are far less than for digoxin (247).

The latter difference is reflected in the recommended oral (or sublingual) dosage (6 -12 mg for ouabain vs. 0.375- 0.5 mg for digoxin) (84, 106, 107).

Ouabain was therefore recommended primarily for acute iv administration in heart failure emergencies with an oral digitalis preparation then used to maintain the cardiotonic effect (84).

Ever since 1885 when Fraser popularized it as a cardiotonic agent (82, 204) some physicians have advocated use of oral ouabain (85).

Digitalis preparations are, however, far more widely prescribed, and ouabain is no longer approved for clinical use in the US, perhaps because iv EO may cause sudden death (3). [ After all, ouabain has been used as an arrow poison for centuries because of its proven efficacy (215)!]


Ouabain's apparently narrow therapeutic window and its low and variable absorption rate are fundamental concerns.

Moreover, there has been no controlled clinical trial of oral ouabain efficacy; all evidence is anecdotal.

To address these concerns we need: 1) a convenient EO assay; 2) solid information about the normal plasma EO level under a variety of conditions (as noted, EO is normally elevated during intense exercise and during pregnancy); and 3) a well-con- trolled clinical trial including plasma EO measurements before and during therapy.


This article summarizes the origins and development of ideas and accrual of knowledge about a newly recognized endocrine system that has been hidden in plain sight.

During the past 25 years numerous ground-breaking discoveries about Na ϩ pumps and CTSs, and their influences on cardiovascular, renal, and brain physiology and pathophysiology have helped to elucidate the EO-Na ϩ pump endocrine system.


The mam- malian Na ϩ pump is a most unusual hormone receptor because it functions both as a cation transporter that does not require the hormone for activity (although some isoforms can be modu- lated by EO), and as an EO-activated signal transducer that appears to function independently of the cation transport.

The ␣-subunit is actually an ancient protein: it is found in Archea and bacteria as well as eukaryotes.

Indeed, hydra, nematode, and fruit fly NKA ␣-subunits have substantial sequence ho- mology and tertiary structure similarity to the human NKA ␣ (208, 222, 281).

The invertebrate ␤- and human ␤-subunit sequences are, however, more distinct (222).

Nevertheless, ouabain also inhibits the hydra and nematode NKAs (41, 51, 52).

Thus, CTS binding activity has been conserved through evolution despite the occasional appearance of ouabain resis- tant pumps, as in rodents and insects (51), but the significance of CTS binding in invertebrates and lower vertebrates is unknown.


It should be very instructive to learn when and how the endogenous CTS-Na ϩ pump couple evolved into a hor- mone-receptor complex.

When, during evolution, did EO first appear? And when did EO-activated cell signaling evolve?


The functional consequences of interfering with the EO-Na ϩ pump (hormone-receptor) interaction, either by mutation of the C16 THE PUMP, THE EXCHANGER, AND THE HOLY SPIRIT AJP-Cell Physiol • doi:10.1152/ajpcell.00196.2017 • www.ajpcell.org Downloaded from journals.physiology.org/journal/ajpcell at McGill Univ (132.216.240.244) on May 18, 2024.


ligand binding site to an ouabain-resistant form or by immuno- neutralization of EO, demonstrate that EO and Na ϩ pumps are an endocrine/paracrine system with very broad impact.

The idea that an endogenous Na ϩ pump inhibitor/CTS is a vaso- tonic agent that plays a role in the pathogenesis of hypertension has been validated (31), but the mechanism is far more com- plicated than originally envisioned (29).

This is exemplified by the multitude of unexpected, dogma-defying findings (see Table 1).

These include: cloning of the Na ϩ pump ␣ and ␤ subunit isoforms; discovery that hormone ouabain (EO) is synthesized in the brain as well as the adrenals, and that circulating EO is regulated by a novel neurohumoral pathway in the hypothalamus; discovery that EO and brain Na ϩ pumps modulate behavior and learning; identification of the ouabain- Na ϩ pump interaction as an activator of PK cascades and cell signaling; recognition that the actions of ouabain-like and digoxin-like CTSs are not identical, and that DLCs do not activate PK cascades; discovery that ouabain/EO can be both pro- and anti-inflammatory; and recognition that EO is a stress hormone and a growth factor and is essential for normal fetal development.

It is difficult to grasp the full scope of this novel humoral system with all of its complexity and myriad actions.


The past 40 years have been an exciting roller coaster ride, but our odyssey is far from complete.

We undoubtedly will learn much more about EO as a hormone when a specific antagonist becomes available, as happened with the RAAS when ARBs like losartan were developed.

We also need a convenient commercial assay for EO.

There are still numerous questions to answer and controversies to resolve, starting with elucidation of the steps in the synthesis of EO from progester- one.

A major concern is whether funds will be available for the requisite studies that utilize standard methods and are not very "glitzy." Because of a research funding squeeze the US is starting to lose its leadership in the EO-Na ϩ pump field despite its obvious clinical relevance, just as research on this novel endocrine system is picking up in Canada, Israel, Italy, Japan, Sweden, and the UK.


The EO-Na ϩ pump system influences the physiology of most, if not all, organs in the body.

The complexity of this system is illustrated by the role of EO and Na ϩ pumps in hypertension (Fig. 4), from whence I began with a far more simplistic view (Fig. 1).


Too often we lose sight of Page's prescient insight that hypertension is a mosaic (230).

Multiple factors (genetic, environmental, immunologic, renal, neural, endocrine, cardiac, and vascular) are essential contributors to the BP elevation- even when a single cause such as a mono- genic defect in salt transport (177) can be identified as the initiating element.


A plethora of components and interacting pathways that influence BP have been elucidated, but they are generally studied one bit at a time, and they can't all be "essential." Which ones are really important? It appears that we need a new, more quantitative approach, a new way to juxtapose all the pieces to determine how these elements all work together to regulate BP.


It's time for the engineer- biologists: we need to learn how to "fix a radio" (164).

Here is the essence and future of (integrative) physiology!


The history of progress with the EO-Na ϩ pump endocrine system calls to mind T. H. Huxley's admonition (letter to C. Kingsley, 1860): "Sit down before fact as a little child, be prepared to give up every preconceived notion, follow humbly wherever and to whatever abysses nature leads, or you shall learn nothing."- but be sure of the "facts."


TO THE EDITOR: In a recent article [2], Dr. Blaustein reiterates his hypothesis that a compound found in some plants—ouabain—is identically present also in human circulation, originating from putative endogenous sources.

In Table 2A of his article, Dr. Blaustein cites previous articles on several spectroscopic studies.

In these studies, various mammalian materials were submitted to complex sample preparation procedures, in part including chemical derivatization to obtain mass or NMR spectra.

These spectra were reported compatible with the presence of ouabain in the sample materials.

While acceptable for generating hypotheses, these data are far from unequivocal identification of a compound.

The main hypothesis generated from this previous work was that ouabain circulates in human blood in concentrations that make a biological function likely.

At this point, a hypothesis generated based on qualitative spectroscopic data had to be confirmed by highly accurate and thoroughly validated quantitative spectrometric data.


Atmospheric pressure ionization coupled to triple-stage mass spectrometry (API-MS/MS) is for more than two decades now the technology of choice for the sensitive and specific quantification of well-defined target analytes in complex biological matrices.

The technology is widely available in research institutions worldwide now and state-of-the-art in quantitative biomedical analytics.

Ouabain pure compound preparation is commercially available.

It is an ideal target for API-MS/MS analyses with good ionization properties.


Based on the reports by Blaustein and others, we decided in 2013 to develop a stable-isotope dilution API-MS/MS method for the quantification of ouabain in human plasma.

We were able to develop a method of extremely high sensitivity detecting spiked ouabain in human plasma.

The method was fully validated according to FDA guidelines and published in the official journal of the International Federation of Clinical Chemistry after peer review [1].

Using this method, we did not detect ouabain within the calibration range of the method (1.7–172 pmol/l) in any of the analyzed unspiked human plasma samples.


In his recent review, Dr. Blaustein now declares [2] a very minor region of increased background noise in a MS/MS-chromatogram from our work [4] with a clearly distinct retention time compared with ouabain to represent a putative isomer of ouabain (footnote to Table 2).

From an analytical perspective this idea is abstruse.

It is beyond all standards of biomedical method validation and scientific work to declare that noise in a published graph of a chromatogram, which is below any reasonable peak detection level, represents a putative compound.


Dr. Blaustein bases his assumption on the existence of an ouabain isomer with profoundly different chromatographic properties eluting as a putative peak mainly on a report by Jacobs et al. [ 3].

In this report no human material was investigated.

An MS spectrum of a fraction extracted from rat plasma sharing some signals with those found in ouabain pure compound is displayed (Fig. 4 of Ref.

[ 3]).

No potential conformational structure of that compound is suggested or discussed.


It is claimed that applying “massive averaging” quantification was achieved.

However, there is absolutely no data reported documenting critical state-of-the-art biomedical method validation.

Essential information on how these experiments could be repeated in independent laboratories is not given.


As long as the presence of ouabain from endogenous sources in human blood is not verified by using quantitative state-of-the-art MS technologies [i.e., API-triple-stage tandem mass spectrometry (MS/MS)] with reproducible quantification, applying rigorous method validation by independent bioanalytical laboratories, the hypothesis that “ouabain immunoassays” actually detect endogenous ouabain in human blood cannot be assumed correct.


In his Letter to the editor (LTE) of AJP-Cell Physiology, Dr. Vogeser [13] raises questions about the identification of endogenous ouabain (EO) in human plasma by mass spectroscopy (MS) and NMR spectroscopy from multiple laboratories including ours.

His laboratory reported that they were unable to detect EO in human plasma [1], but serious concerns were raised in my Letter to the Editor of that journal [3] and in a subsequent article [7].


The basis for the discrepant (negative) results reported in Ref.

1 is unclear; however, the only data are contained in Fig. 2 of Ref.

1, which shows a plasma sample spiked with authentic ouabain.

Thus, any endogenous ouabain would have been obscured.

Moreover, while Vogeser and colleagues reportedly tested “30 human plasma samples” [1], no data are presented for any of the other 29 samples and only one other sample is shown in their subsequent LTE [12].

Below I address the technical issues raised by Vogeser.


Regarding the spectroscopic evidence for EO cited in my Table 2A [4], Vogeser criticizes the fact that “various mammalian materials (plasma, adrenal cortex, adrenal cells and conditioned media, hypothalamus, and urine) were submitted to complex sample preparation procedures including chemical derivatization to obtain mass or NMR spectra.

” This statement confuses several issues and is misleading.

First, chemical derivatization was used in only a few instances.

Second, in most cases, multiple separation steps were required to isolate EO in the purity and amounts sufficient for outright identification.

The fact that these isolates, from human, bovine, and rodent sources, treated in so many different ways, and analyzed by both MS and NMR (e.g., see Refs. 10, 11), were all found to contain the same compound (EO) is remarkable and belies Vogeser’s concerns.

In several studies, three-dimensional MS (MS-MS-MS) collision-induced dissociation revealed ion spectra identical to those of authentic ouabain before and after dissociation of the sugar and, more significantly, after fragmentation of the steroid moiety [7-9].

Also, in several of the reports in Table 2A [4], as well as in other studies of human plasma and adrenal cell-conditioned media lacking spectral confirmation and therefore not included in Table 2A (e.g., Refs. 2, 5, 6), ouabain-like compounds (OLC) were detected by bioassay (e.g., Na+-K+-ATPase inhibition) both before and after the separation steps.


The OLC in all these studies co-eluted with authentic ouabain.

Finally, bovine adrenocortical cell secretion of the OLC (identified by bioassay, NMR, and MS), but not cortisol, was increased by incubating the cells with rhamnose, suggesting that the OLC, like authentic ouabain, was a rhamnoside [10].

Together, these multifarious studies provide compelling evidence that the endogenous compound is, in fact, ouabain.


In his LTE, Vogeser’s disparagement of “massive averaging” [9] of the MS-MS and MS-MS-MS spectra reflects a misunderstanding.

Baecher et al. [ 1] interrogated the output of their ultraperformance liquid chromatography (UPLC) directly with online MS-MS.

In this type of liquid chromatography (LC)-MS-MS, the LC runs are so rapid that the mass analyzer only has time (~6 s) to target one or two product ions and generate a single spectrum.

The primary target in Ref.

1 was a single dominant product ion whose m/z (mass/charge) ratio was equivalent to ouabagenin’s.


In contrast, Jacobs et al. [ 9] collected high-pressure LC (HPLC) fractions, and used radioreceptor and radio-immunoassays to identify the fractions that were “ouabain-reactive.

” Those fractions were then interrogated, offline, by MS-MS and MS-MS-MS.

The offline approach allows the mass analyzer long periods of time (up to several minutes) to generate and average a very large number of replicate spectra from each sample (i.e., “massive averaging”).

This not only yields high-quality spectra with minimal noise but, in the case of MS-MS-MS, gives detailed information about multiple prominent fragments of the steroid moiety (e.g., Fig. 4C in Ref.

9 and Supplementary Information in Ref.

8) that cannot be readily obtained by LC-MS-MS instruments.


Vogeser’s implication that the “hypothesis based on (these) qualitative spectroscopic data” is invalid without precise quantitation of the ouabain level is unjustified and misleading.

The ion current peaks from the offline MS-MS-MS can be quantitatively compared with ouabain and, when required [8], compared directly with an internal standard (dihydroouabain).


Contrast nearly all the OLC studies referenced above [2, 4-11], in which bioassay (Na+-K+-ATPase binding or inhibition) was used to verify the presence of OLC, with that of Vogeser [1]: There is no indication of an effort to determine whether their native plasmas contained any bioassay- or immunoassay-positive OLC; their only measurement was MS [1].

Their sample treatment included [1]: dilution with 0.1% trifluoroacetic acid, centrifugation, solid phase extraction and evaporation to dryness of the supernate, reconstitution in 5% methanol, and UPLC with online MS-MS.

Calibration consisted of MS analysis of “a pool of EDTA plasma from leftover samples (spiked) with a solution of (plant) ouabain in methanol 20%” [1].


No original recordings of the calibration or unaltered sample spectra were shown in Ref.

1. Their “method was fully validated” [1], presumably for other steroids, but not optimized for OLC even though ouabain is an unusual, polar steroid.

For example, why reconstitute samples in 5% methanol, the solvent used to elute ouabain, when ouabain is water soluble and should stick to the LC column better when dissolved in water than in 5% methanol (Ref.

7 data supplement)?

All the “spiked plasmas” and calibration samples involved a step that was different from the native plasma alone.

Could the reconstituted samples have contained an OLC that did not stick to the UPLC column, and was washed through before applying the very steep UPLC water-methanol gradient, starting at 5% methanol?

Note that the spectrum in Fig. 2 [1] begins at 3.0 min—close to 35% methanol in the gradient.

Because of the very rapid (~6 min), steep LC gradient runs, it would be surprising if everything stuck to the column and there was no ion current or even “noise” before 3.0 min.


In summary, Vogeser’s attempt to use a more sensitive MS-MS system to quantify EO levels is commendable.

However, while his MS system may be “state-of-the-art,” if the endogenous compound is lost before the MS analysis, the most sensitive MS system will not detect it.

The shortcomings of the Baecher et al. study [1] should not detract from the numerous reports that EO is ouabain, that it circulates in human plasma, and that it is a mammalian hormone.


The aim of this review is to provide current knowledge on fertility preservation for non-medical reasons in women willing to postpone childbearing.

The topic is highly debatable, starting from disagreement about its terminology, the number of eggs necessary to predict chances of success, and the safety and socio/ethical point of view.

Cost analysis and discrepancies among countries' recommendations and regu- lations are described to confirm the controversies and unsolved issues around this very interesting topic.

Finally, an overview on the returning rate of women among "egg bankers" and reasons behind their decisions are illustrated.


In early 2012, ESHRE embraced the idea of oocyte cryopreservation for age-related fertility loss, recommending the need of adequate information for women interested in postponing childbearing with- out creating false hopes.

12 One year later, instead, both ASRM and Society for Assisted Reproductive Technology (SART) argued that oocyte cryopreservation should not be recommended in order to bypass the age-related fertility decline due to the absence of data supporting safety, efficacy, ethics, emotional risks and cost-effec- tiveness of egg freezing for that indication.

11 Subsequently, the American College of Obstetricians and Gynecologists (ACOG) en- dorsed the guideline of ASRM-SART.

18 The Canadian Fertility and Andrology Society (CFAS) produced guidelines to manage this type of patient, highlighting the need to counsel women about their chances of success.

19 Recently, the Royal College of Obstetricians and Gynaecologists (RCOG) suggested caution in the case of women who wish to cryopreserve their oocytes for social reasons, because cryopreserved oocytes do not guarantee a child in future.

Moreover, in the UK, the length of cryostorage of oocytes for non-medical rea- sons is currently limited to 10 years.


It is well known that the success rate of in vitro fertilization (IVF) cycle using cryopreserved oocytes declines rapidly as the age of a woman increases.

21 A retrospective observational multicen- tric study evaluated 1468 women undergoing elective oocyte cryopreservation due to age-related fertility decline (n = 1382) or medical conditions other than cancer (n = 86).


In women aged ≤35 years, the cumulative live birth rate (CLBR), considering the total number of oocytes used in consecutive procedures, was sig- nificantly higher compared with those aged >36 years, despite the same number of oocytes being utilized.

22 Moreover, stratifying by age, that study showed an appreciable decrease in live birth rate from the youngest category of women aged ≤29 years (100%; 95% confidence interval [CI] 100-100) to the oldest group of women aged 40-44 years (3.7%; 95% CI −3.4 to 10.8).

22 Other studies reached a similar conclusion, showing that the highest probability of live birth from cryopreserved oocytes is obtained before the age of 36-38.

23,24 Another study based on a decision analysis model cor- roborated that at the age of 37 there is the largest benefit in terms of probability of live birth compared with no action.

25 Despite that, Tsafrir et al 26 recommended that the optimal age for oocyte cryo- preservation should be, preferably, before 35 years of age.


These data indicate the lack of agreement regarding the optimal timing of oocyte cryopreservation.


Unfortunately, most women undergo treatment after a significant decline in fertility.

Indeed, many stud- ies based on surveys, showed that average ages to cryopreserve oocytes are between 36 and 38 years.

14,15,27,28 Therefore, proper counseling must ensure that women are aware of the fact that their frozen oocytes do not represent an insurance policy against age-re- lated infertility, but rather the payment for an extra opportunity.


It is worth mentioning that, among all factors influences the probabil- ity of achieving a live birth using cryopreserved oocytes, the num- ber of retrieved mature oocytes is of the highest importance.

Indeed, freezing 8 or 10 oocytes gave a 40.8% or 60.5% probability of a live birth per warmed oocyte, respectively, for women under the age of 35.

22 In women older than 35 years who used 8 or 10 oocytes, the CLBR decreased to 19.9% and 29.7%, respectively.

22 In women aged ≤35 years, the increase of CLBR appears slightly slower from 10 to 15 oocytes, obtaining a maximum CLBR of 85.2%.

Instead, in older women, the CLBR plateau of 35.6% is reached with 11 frozen oocytes, indicating that success was not increased notwithstanding the in- crease in the number of oocytes.

22 Regardless of the suggestion from Cobo et al of a minimum of 8-10 cryopreserved oocytes to obtain a realistic chance to achieve a pregnancy, another study estimated that women between 35 and 38 years of age with 20 cryopreserved oo- cytes would have live birth rates of 80% and 60%, respectively.

24 Recently, a mathematical model has been developed to predict the probability of live birth based on the number of cryopreserved oocytes taking into consideration female age.

30 According to this model, women at 34, 37 or 42 years would need to cryopreserve 10, 20 and 61 oocytes, respectively, to obtain a 75% likelihood of having at least one live birth.

30 Although this model could represent an interesting counseling tool, the limitation of this study is the sole inclusion of women with normal ovarian reserve undergoing intra- cytoplasmic sperm injection due to male factor or tubal factor in- fertility.

It is important to explain to these women that, to obtain an adequate number of oocytes, multiple cycles of controlled ovarian stimulation might be needed.


The most important medical risks associated with oocyte cryo- preservation are associated with ovarian stimulation and oocyte


Oocyte cryopreservation is an established and widely used treatment for women who are willing to postpone child- bearing.

Women must be counseled about realistic success rate and on the fact that preserving oocytes do not repre- sent an insurance policy against age-related fertility decline.


Moreover, a most common consequence of ovarian stimulation is ovarian hyperstimulation syndrome (OHSS).

Clinically, OHSS can be mild, moderate or severe/critical.

Mild and moderate OHSS are equally common, with an incidence between 3% and 6%, and is characterized by abdominal pain, enlarged ovaries and weight gain.

These adverse effects can be well managed.


Conversely, severe/critical OHSS occurring in 1%-3% of IVF cycles involves as- cites, pleural effusion, massive ovarian enlargement, oliguria, hemo- concentration and thromboembolic phenomena up to deep venous thrombosis and embolism.

Severe/critical OHSS is potentially life- threatening and most worrisome.


Luckily, due to the implemen- tation of ovarian stimulation protocols using antagonist with agonist trigger, the risk of developing OHSS is very reduced, although not yet eliminated.


It is relevant to consider that elective oocyte cryopreservation for non-medical reasons does not prevent maternal morbidity in women who become pregnant at an older age.

In fact, the pregnancy in older women is associated with a higher risk of ectopic pregnancy and other complications such as preeclampsia, gestational diabetes, preterm delivery and low birthweight.

37 For this reason, appropriate counseling of women cryopreserving oocytes, regarding the poten- tial risks of pregnancy at an advanced age should be mandatory.

It is reassuring that, so far, no relevant obstetric and perinatal risks were found in pregnancies achieved with frozen oocytes, confirming that oocyte cryopreservation does not seem to be a harmful proce- dure.

38-40 Moreover, long-term cryopreservation of oocytes in liquid nitrogen does not affect the IVF success outcomes or the euploidy rate.

41,42 No studies as yet have reported long-term follow up of chil- dren born following oocyte cryopreservation.


There are few data about the percentage of women who return to use their frozen oocytes.

This parameter is called the "usage rate", and represents a critical issue for egg freezing for non-medical in- dications.

Four studies based on surveys have analyzed follow-up data of "bankers".

14,15,27,28 One of these demonstrated that only 50.8% of women who cryopreserved oocytes thought they would use their eggs in the future.