Prorenin anno 2008

For many years, prorenin has been considered to be nothing more than the inactive precursor of renin. Yet, its elevated levels in diabetic subjects with microvascular complications and its extrarenal production at various sites in the body suggest otherwise. This review discusses the origin, regulation, and enzymatic activity of prorenin, its role during renin inhibition, and the angiotensin-dependent and angiotensin-independent consequences of its binding to the recently discovered (pro)renin receptor. The review ends with the concept that prorenin rather than renin determines tissue angiotensin generation.
Introduction
Despite nearly 40 years of research on prorenin, the renin precursor is still the least well-understood component of the renin–angiotensin system (RAS). Initially, it was thought to have no function at all. Yet, it circulates in human plasma in excess to renin, sometimes at concentrations that are 100 times higher, and in plasma of anephric subjects, prorenin, but not renin, is still present [1, 2]. This suggests that prorenin, in contrast with renin, is also of extrarenal origin. Prorenin is particularly elevated in diabetic subjects with microvascular complications [3]. Moreover, the renal vasodilator response to captopril in diabetic subjects correlated better with plasma prorenin rather than with plasma renin [4]. Thus, (circulating) prorenin may have a function after all. Possibly, it is prorenin (and not renin) which is responsible for tissue angiotensin generation. Obviously, this would require local prorenin–renin conversion, for which no evidence exists [5]. In support of this concept, however, transgenic rodents with (inducible) prorenin expression in the liver display increased cardiac angiotensin (Ang) I levels, cardiac hypertrophy, and/or vascular damage [6, 7].
Origin and regulation of prorenin
The juxtaglomerular epithelioid cells, located in the walls of renal afferent arterioles, are the main source of renin in the body. Renin is synthesized as preprorenin. Preprorenin is converted to prorenin upon insertion into the endoplasmatic reticulum. The majority (75%) of prorenin is secreted constitutively, while the remainder is targeted to dense core secretory granules. In these granules, an acidic pH is created to optimize the activity of the proteases (cathepsin B, prohormone convertases) that cleave off the prosegment to yield renin. Prorenin and renin levels are highly correlated but do not alter in parallel under all circumstances [1]. Acute stimuli of renin will not affect prorenin levels, whereas chronic stimuli (like a decrease in Ang II) increase both renin and prorenin. This suggests that renin is stored as active enzyme and is released immediately upon stimulation of the juxtaglomerular apparatus. Prorenin is released constitutively, and no acute responses occur. Chronic stimulation causes more prorenin to be converted to renin, leading to an increased renin/prorenin ratio in plasma. However, some exceptions to this rule exist. A well-known example is, as mentioned above, diabetes mellitus complicated by retinopathy and nephropathy [3]. Pregnant women also have high plasma prorenin levels, derived from the ovaries [8]. The function of this prorenin is unknown, as is the function of prorenin in amniotic fluid, in which prorenin was discovered. The reproductive organs, together with the adrenal, eye, and submandibular gland, are sites of extrarenal renin gene expression [9]. For reasons that are not understood, these tissues predominantly, if not exclusively, synthesize and release prorenin.
Enzymatic activity of prorenin?
A 43-amino-acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. Prorenin can be activated in two ways: proteolytic or nonproteolytic [10]. Proteolytic activation is irreversible: it involves actual removal of the propeptide. Nonproteolytic activation of prorenin is reversible. It can best be imagined as an unfolding of the propeptide from the enzymatic cleft. Nonproteolytic activation can be induced by exposure to low pH (pH = 3.3) or cold (4°C) [10]. Nonproteolytically activated prorenin is enzymatically active and can be recognized by monoclonal antibodies that are specific for the active site. Kinetic studies of the nonproteolytic activation process have indicated that an equilibrium exists between the closed (inactive) and open (active) forms of prorenin. The inactivation step is highly temperature dependent and occurs very rapidly at neutral pH and 37°C. Consequently, under physiological conditions, <2% of prorenin is in the open and active form, i.e., displays enzymatic activity, and >98% is closed and inactive.
Prorenin receptor(s)?
The beneficial effects of RAS blockers are due, at least in part, to blockade of the generation or action of Ang II at tissue sites [11]. In tissues that are believed not to express the renin gene, like the heart and vascular wall, such angiotensin generation depends on renin/prorenin taken up from the circulation. Simple diffusion cannot explain the relatively high renin levels in these organs, and thus a receptor-mediated mechanism may exist. Two candidates currently have been proposed: the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) [12–14] and the (pro)renin receptor [15]. The M6P/IGF2R nonselectively binds M6P-containing proteins like renin and prorenin. However, such binding did not result in angiotensin generation, and it is now believed that the M6P/IGF2R is a clearance receptor for renin/prorenin [16]. This leaves the (pro)renin receptor as the most promising candidate for tissue uptake of circulating renin/prorenin. This receptor, a 350-amino-acid protein with a single transmembrane domain, binds prorenin with higher affinity than renin [17] and, unlike the M6P/IGF2R, does not internalize these proteins. Interestingly, binding to the receptor allowed prorenin to become catalytically active without proteolytic cleavage of the prosegment [15, 17]. Apparently, therefore, binding induces a conformational change in the prorenin molecule, similar to the change occurring after exposure to cold or low pH.
Angiotensin-independent effects of prorenin?
After the discovery of the receptor, (pro)renin receptor antagonists were designed based on the idea that the prosegment contains a ‘handle region’ which binds to the receptor [18]. These (peptidic) antagonists (also known as ‘handle region peptides’, HRP) mimic the handle region and thus may bind to the receptor instead of prorenin. In support of this concept, HRP infusion normalized the elevated renal angiotensin content in diabetic rats [18] and simultaneously prevented the development of diabetic nephropathy.
Surprisingly, identical effects occurred in diabetic angiotensin II type 1A receptor-deficient mice [19]. Since such mice no longer display the normal (constrictor) response to Ang II [20], the effect of the (pro)renin receptor antagonist in these mice cannot be due to suppression of local angiotensin generation. Thus, prorenin may also exert direct angiotensin-independent effects, possibly via the above described (pro)renin receptor (Fig. 1). Indeed, prorenin (and renin) induced p42/p44 mitogen-activated protein kinase (MAPK) activation and transforming growth factor-β1 release in mesangial cells [15, 21], and these effects did not occur following deletion of the receptor with siRNA [21]. Moreover, in cardiomyocytes, prorenin concentration dependently activated p38 MAPK and phosphorylated heat shock protein 27 [22].
Fig. 1Model depicting prorenin activation by the (pro)renin receptor (P)RR, allowing prorenin to generate angiotensin I from angiotensinogen. In addition, prorenin binding to the receptor results in effects (intracellular signaling) that are independent of angiotensin generation. HRP is assumed to block both prorenin activation and the direct prorenin-induced effects (see text for further explanation)
Overexpression of the human (pro)renin receptor in rats resulted in elevated blood pressure, increased plasma aldosterone, and/or glomerulosclerosis [23, 24]. Since such overexpression was not accompanied by changes in renin or Ang II, angiotensin-independent effects of the receptor may underlie this phenotype. HRP prevented the development of glomerulosclerosis in (pro)renin receptor transgenic rats [25]. Yet, transgenic rats with inducible hepatic prorenin expression (resulting in a >200-fold rise in plasma prorenin) did not develop glomerulosclerosis [26], although such animals did develop hypertension. Moreover, HRP blocked neither prorenin binding to cells overexpressing the human (pro)renin receptor [17] nor prorenin-induced signaling in U937 monocytes [27]. Thus, it is uncertain to what degree the beneficial in vivo effects of HRP are solely due to prorenin blockade. Such effects are unlikely to involve renin, since HRP will not block renin–(pro)renin receptor interaction.
Prorenin and renin inhibition
Prorenin levels will rise during renin inhibition, as they do during any type of RAS blockade. Renin inhibitors affect the equilibrium between the open and closed forms of prorenin, because such drugs (due to their high affinity for the active site) prevent inactivation [10]. Therefore, renin inhibitors, like low pH, cold, and the (pro)renin receptor, are capable of nonproteolytically ‘activating’ prorenin although, of course, due to the presence of the renin inhibitor, this open prorenin cannot display enzymatic activity. Open prorenin will however be recognized by the active site-directed antibodies applied in renin immunoradiometric assays, thus leading to an overestimation of the renin rise during renin inhibition [28].
Theoretically, the elevated (pro)renin levels during renin inhibition might result in (pro)renin receptor activation. In vitro studies suggest that renin inhibitors do not interfere with this process. The consequence of such overstimulation is unknown. However, since such a rise also occurs during other types of RAS blockade, its detrimental effects, if present, should have been known by now. A possible explanation comes from the work of Schefe et al. [29], who showed that, on activation of the receptor, the transcription factor promyelocytic zinc finger is translocated to the nucleus and represses transcription of the (pro)renin receptor itself, thus creating a short negative feedback loop. In other words, high (pro)renin levels, as occurring during RAS blockade, will suppress (pro)renin receptor expression, thereby preventing excessive receptor activation. Furthermore, the members of a family with a mutated renin allele, resulting in high plasma prorenin levels, were phenotypically normal [30].
Conclusion
After many years, it now seems that a function for prorenin has been found. The ‘inactive’ renin precursor gains Ang I-generating activity by binding to a receptor, without undergoing proteolytic cleavage. This mechanism explains how prorenin might contribute to tissue angiotensin generation, even when no prorenin–renin conversion occurs outside the kidney. Renin inhibitors will bind to such open activated prorenin, to the same degree as they bind to renin, and may thus be the ideal tools to block tissue angiotensin generation. In vitro studies suggest that prorenin also acts as an agonist of the (pro)renin receptor, inducing intracellular signaling pathways in an angiotensin-independent manner. However, given the contradictory data obtained with the (pro)renin receptor blocker HRP, more work is needed to verify the in vivo importance of such prorenin-induced (pro)renin receptor activation.

renin
diabetes
(pro)renin receptor
receptor
tissue angiotensin
signaling
renin inhibitor
renin angiotensin system


Viscosity Is Not a Parameter of Postdeglutitive Pharyngeal Residue: Quantification and Analysis with Scintigraphy

The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers. Scintigraphy was used to measure pharyngeal residue in 11 healthy volunteers after swallowing three different substances (age = 20.2–48.3 years). The first substance was a 10-ml solution of tap water with 0.5% xanthan with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were a 0.75% xanthan and a 1.00% xanthan solution, with viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. Tap water was used as the control substance. Mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. Pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution 2.8% (SD = 1.7). No significant correlation between increase of viscosity and pharyngeal residue was found. In healthy persons viscosity does not seem to be a significant parameter for pharyngeal residue for boluses with viscosities ranging from tap water to solutions having a viscosity comparable to 3% fat yogurt.
The residual amount of food or beverage in the pharynx after swallowing (pharyngeal residue) is an important parameter in diagnostic procedures in the treatment of swallowing disorders. In common practice of management of swallowing disorders, the amount of pharyngeal residue is determined either by videofluoroscopy or by transnasal flexible endoscopy (FEES). With the help of logistic regression, Perlman et al. [1] predicted the chance of aspiration (food or liquid that gets into the lungs) based on the amount of pharyngeal residue. An odds ratio of 1.4 for aspiration was found for patients with pharyngeal residue. In this study 330 videofluoroscopies of dysphagic patients were analyzed and the amount of pharyngeal residue was rated on a four-point scale (absent, mild, moderate, severe). The study also showed that a patient with severe pharyngeal residue has an increased risk of aspiration (odds ratio of 4.0) and underlined the importance of quantifying pharyngeal residues. Dejaeger et al. [2] analyzed videofluoroscopies of 25 healthy elderly and clearly described localization of pharyngeal residue; unfortunately, they did not attempt to quantify the amount of residue.
Few attempts have been made to quantify pharyngeal residue in normal and dysphagic populations, mainly because of the limited availability of appropriate diagnostic tools. Videofluoroscopy or FEES has been used to explore physiologic parameters of swallowing [3,4]. One of the limitations of the use of these techniques is the lack of reliable interpretation of physiologic parameters. Stoeckli et al. [5] analyzed the inter- and intrajudge reproducibility of the interpretation of videofluoroscopy and suggested that the agreement for assessing pharyngeal parameters is minimal to moderate (κ = 0.01–0.56).
Scintigraphy allows quantitative measurement by using a technetium-99m (99mTc)-labeled colloid. 99mTc colloid is used in routine clinical practice for investigating gastrointestinal motility disorders of the esophagus and stomach [6,7]. The scintigraphic technique allows quantitative assessment because the amount of radioactivity (radioactive counts) can be measured with a gamma camera. Scintigraphy in dysphagia mainly is used for detection of aspiration in young children because the radiation doses are less than those of videofluoroscopy [8]. It is used less frequently in adults, although recently it has been described as a useful and reliable diagnostic tool in adult patients with oropharyngeal dysphagia [9]. Only three studies have been performed with scintigraphy to examine pharyngeal parameters of normal swallowing physiology: Cook et al. [10] used scintigraphy to study the influence of age on swallowing efficiency. Hamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers, while Shaw et al. [12] studied the influence of bolus volume on pharyngeal residue. All these studies examined different parameters of swallowing and found different outcomes. The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers, where our hypothesis was that swallowing a more viscous product would result in more pharyngeal residue in healthy volunteers.
Methods
Volunteers
Eleven healthy volunteers (9 female, 2 male, mean age = 29.1 years, standard deviation [SD] = 8.9), recruited by public advertisement, were invited to participate. All volunteers reported no history of swallowing problems. All volunteers gave written informed consent to participate in the study, which was approved by the Medical Ethics Committee of the Academic Medical Center of the University of Amsterdam.
Bolus Characteristics
Before the examination, three different substances with three different viscosities were prepared in a research laboratory. All viscosities were measured with a viscosity meter (Brookfield LVT Viscometer, model DV-II+) after 24 h of storage in a refrigerator at a constant temperature of 4°C. Measurements were performed with spindle No. 63 at a velocity of 3 rpm and viscosities were recorded after 30 s. The substances consisted of tap water thickened with different percentages of xanthan, a polysaccharide commonly used in the food industry to thicken fluids. The first substance was a solution of tap water with 0.5% xanthan resulting in a substance with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were tap water with 0.75% and 1.00% xanthan, resulting in viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. As reference, tap water with a viscosity of 1 mPa s (per definition) was used. For all solutions 200 ml was labeled with 260 MBq of 99mTc colloid (Hepatate, GE Health, Eindhoven, The Netherlands) dissolved in 1 ml 0.9% saline solution and prepared in such a way that for 10 ml of each solution the radiation dose was 13 MBq. After labeling, 44 syringes (4 per volunteer) were used to collect 10 ml of each of the four solutions (tap water, 0.5%, 0.75%, and 1.00% xanthan). The three xanthan solutions, stored for more than 24 h in a refrigerator at a stable temperature of 4°C, were mixed with the 99mTc colloid 1 h before the study.
Data Collection
For this study a gamma camera (Orbiter, Siemens Medical Systems, Germany) was fitted with a low-energy all-purpose collimator. A dynamic acquisition using 0.25-s frames was performed for 30 s. Data were acquired and processed on a Hermes workstation (Nuclear Diagnostics, Sweden). Volunteers were asked to sit in front of the gamma camera and hold their right cheek against the collimator. The oral cavity was placed in the middle of the field-of-view. When the volunteer was placed in the right position, 10 ml of one of the solutions was emptied in the mouth of the volunteer with a syringe. Volunteers were instructed to hold the bolus in their mouth and swallow on command. They were specifically instructed to swallow the whole bolus in one swallow and to breath through the mouth to inhibit a second swallow. Three cobalt markers were placed on the skin of the volunteers using a pen pointer: one on the mastoid bone, one on the hyoid bone, and one underneath the thyroid cartilage. The exact placement for the marker was found by palpation of the head and neck.
First, the volunteers were asked to swallow the water with 99mTc colloid. After recording the swallow for 30 s, the volunteers were asked to rinse their mouth with unlabeled tap water and to swallow 200 ml unlabeled tap water to wash away any residue from the previous recording. The second recording was done with 10 ml of the 0.5% xanthan solution, the third with the 0.75% xanthan, and the last with the 1.00% xanthan. Between recordings the volunteers were instructed to rinse their mouth and swallow 200 ml unlabeled tap water to clear any possible residue. Each volunteer swallowed a consistency labeled with a dose of 13 MBq four times, for a total of 52 MBq, which is equal to a radiation dose of less than 1 milliSievert (mSv). According to the ICRP62 guidelines, this is a minor radiation dose (category IIa).
After data collection the results were stored and processed on a Hermes workstation. Based on the markers, two regions of interest (ROI) were defined: the oral cavity (ROI-1), bordered by a straight line between the hyoid marker and the mastoid marker, and the pharynx (ROI-2), bordered by a straight line between the hyoid marker and the mastoid marker and a horizontal line from the thyroid marker, as described by previous studies [10,11]. To estimate background activity during the examinations, a third ROI with a comparable surface area to ROI-2 was defined to the right of the field of view. Figure 1 shows the three ROIs. Collected data were transferred to a spreadsheet file (Excel, Microsoft Corp., Redmond, WA), which showed the number of counts within the specific ROI at each frame (0.25 s) of the total acquisition. For our analysis the average count of 8 frames (2 s) before swallowing was used to determine the average number of counts in the oral cavity. After swallowing the average count of 4 s (16 frames) was used to determine the average amount of pharyngeal residue, expressed in counts. Background activity was estimated by a measurement of 4 s; this was subtracted from the average count in the oral cavity and the pharyngeal residue. Hereafter, the number of pharyngeal counts was divided by the number of counts in the oral cavity, thus providing a percentage of pharyngeal residue.
Fig. 1.Scintigraphic evaluation with three regions of interest (ROI). 1= oral cavity, 2 = pharynx, 3 = background radiation, a = mastoid marker, b = hyoid marker, 3 = thyroid marker.
Statistical Analysis
The calculated percentages of pharyngeal residue were transferred to SPSS v12.0 (SPSS Inc., Chicago, IL) for further analysis. Distribution of the percentages was formally tested with the Kolmogorov–Smirnoff test for normality and was not found to be normally distributed. To explore differences in median percentages of pharyngeal residues of the three xanthan solutions to the reference (tap water) on a group level, a nonparametric (Friedman) test was used. All statistical tests were two-tailed and differences were evaluated at the 5% level of significance.
Results
The mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2; range = 1.1%–4.5%) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing the 0.5% xanthan solution was 1.8% (SD = 0.8; range = 0.9%–2.8%), after swallowing the 0.75% xanthan solution the residue was 2.6% (SD = 2.2; range = 0.7%–8.1%), and after swallowing the 1.00% xanthan solution the pharyngeal residue was 2.8% (SD = 1.7; range = 1.2%–6.4%).
Median percentages of pharyngeal residue were analyzed. Figure 2 shows by boxplot the distribution of residue for the four different viscosities in the 11 nondysphagic volunteers. No significant difference in the amount of residue between the four groups was found (χ2 = 0.927, df = 3, p = 0.819).Fig. 2.Distribution of percentages residue on group level.
Although the sample size was limited, post hoc analysis showed that this study had a power of 0.96 (n = 11 with an effect size of 0.47 based on a SD and correlation between measurements occasions of 1.1 and 0.7, respectively). Based on these results, a power calculation showed that in future research (with a power of 0.80 and alpha of 0.05) at least 137 healthy volunteers would be needed to detect any possible difference in the amount of pharyngeal residue between the four groups (tap water, 0.5%, 0.75%, and 1.00% xanthan) (Table 1).
Table 1.Pharyngeal residue in percentages with 10ml bolusesNo.Water0.5% Xanthan0.75% Xanthan1.00% Xanthan11.11.20.71.221.81.21.01.731.30.78.13.441.41.51.71.354.12.32.66.462.91.51.51.271.20.91.22.482.42.55.35.493.12.52.42.4102.02.52.12.7114.52.82.12.4Mean2.3 (SD ± 1.2)a1.8 (SD ± 0.8)a2.6 (SD ± 2.2)a2.8 (SD ± 1.7)aaNo significant difference (p > 0.05).
Discussion
Our study attempted to investigate the correlations between viscosity and pharyngeal residue in healthy volunteers; we did not find evidence for a positive correlation between an increase of viscosity and an increase in pharyngeal residue.
Because Shaw et al. [12] established that bolus volume does not influence pharyngeal residue, allowing extrapolation of our findings to other volumes, only one bolus volume was used in this study. It might be suggested that our volunteers drank 200 ml tap water after the first labeled bolus and thus the mucosa of the pharynx was relatively dry before the first estimation of pharyngeal residue. Because the larger proportion of our volunteers had drunk coffee, tea, or water in the last hour before the study, differences in dryness of the pharyngeal mucosa is not considered a confounder.
Only one bolus per viscosity was used, therefore no data on intrasubject differences could be provided. If multiple boluses per viscosity had been used in this study, the radiation exposure for our volunteers would have been much higher. Because none of the volunteers had swallowing disorders and all were used to swallowing tap water and yogurt, any adaptation in swallowing during the study to the circumstances during data collection or intrasubject differences can be considered negligible.
Raut et al. [13] examined with manometry the influence of increased viscosity on pharyngeal pressures. It was concluded that an increased bolus viscosity led to increased amplitude of the bolus wave and clearing contractions within the pharynx. The increase in contraction of the pharyngeal muscles supports our findings of equivalent percentages of residue for the different viscosities.
Hamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers. In that study 20 healthy volunteers (age = 39–65 years) swallowed tap water and a thickened substance labeled with 99mTc. The mean pharyngeal residue was found to be 4.0% (standard error [SE] = 0.5) for tap water and 10.5% (SE = 0.1) for a substance with a viscosity of 1070 mPa s.
Differences in the Hamlet et al. study and our study might be explained by temperature influences and different approaches to measure viscosity. In the Hamlet study [11] the bolus temperature was set at 25°C, whereas in our study syringes were stored in a chilled room. Therefore, temperature is not considered a confounding factor in our study. Also, in the Hamlet study [11] a Brookfield Viscometer type RVT was used, which is more suitable for measuring the viscosity of low-viscosity products like milk. In our study we used a Brookfield Viscometer type LVT, which is more suitable for high-viscosity substances, making an adequate comparison between measured viscosities of the two studies not possible.
Literature suggests that body positioning, bolus temperature, and taste affect swallowing [3,4,14]. In our study the volunteers had to rotate their heads to the right side to obtain adequate measurements, following the protocol used in the Hamlet study [11]. This might have reduced the amount of pharyngeal residue because the bolus passes the pharynx mainly on one side and the contact area between bolus and pharyngeal wall is decreased. It is known that in some dysphagic patients (i.e., patients with unilateral pharyngeal weaknesses) rotating the head decreases the amount of residue, but the effects of head rotation on healthy individuals has not been investigated. Looking at the wide distribution of percentages of residues in volunteers (Table 1) and the slightly lower percentage (2.3%) found in our study compared with that of Shaw [12] (2.5%), where measurements were made in a right lateral position, one might conclude that the influence of head rotation in our study cannot be seen as a confounding factor on the outcome.
Conclusion
In 11 healthy volunteers, mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution the residue was 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution the residue was 2.8% (SD = 1.7). Our data do not show a significant increase in pharyngeal residue in healthy volunteers as a result of an increasing viscosity of viscous solutions, ranging from tap water to full-fat yogurt. Although some trends may be seen, (confounding) factors other than viscosity may play a role in the build up of pharyngeal residue.

physiology
deglutition
radionuclide imaging
deglutition disorders


Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.
Introduction
In order to understand carbon partitioning, the regulation of photosynthesis, phloem loading, or sink–source interactions, it is important to know how photoassimilates are distributed between the subcellular compartments of mesophyll cells of source leaves. Much of the information concerning subcellular metabolite concentrations is derived from studies of species such as spinach, barley, tobacco, and potato. In such plant species carbohydrates assimilated from CO2 are exported into the phloem exclusively in the form of sucrose (Riens et al. 1991; Winter et al. 1992; Lohaus et al. 1998). However, it has been shown that in several plant species, sugar alcohols like mannitol, sorbitol or dulcitol are also translocated in addition to sucrose (Zimmermann and Ziegler 1975). The advantage of transporting sucrose and sugar alcohols is that these compounds are highly soluble and chemically inert. In addition to functioning as a transport form for reduced carbon, sugar alcohols have been also shown to serve as storage for reduced carbon, as compatible solute synthesized in response to abiotic or biotic stress, or as osmoprotectants. It has been estimated that up to 30% of the carbon fixed by plants on earth may be present in the form of polyols (Bieleski 1982).
Mannitol is the most-widely distributed sugar alcohol and has been reported in more than 100 species of vascular plants, including most species of the Apiaceae (celery, parsley, and carrot), Oleaceae (olive, privet), Rubiaceae (coffee), and Scrophulariaceae (snapdragon) (Zimmermann and Ziegler 1975; Bieleski 1982). In higher plants, mannitol is synthesized by the action of an NADPH-dependent mannose-6-phosphate reductase that catalyzes the conversion of mannose-6-phosphate to mannitol-1-phosphate, followed by dephosphorylation by a phosphatase (Rumpho et al. 1983). The activity of mannose-6-phosphate reductase was only observed in mature leaves. In different plant species, mannitol synthesis occurs simultaneously with either sucrose synthesis, as in celery (Rumpho et al. 1983), or with raffinose oligosaccharide synthesis, as in Oleaceae (Zimmermann and Ziegler 1975). To date, there are only few reports on the intracellular distribution of mannitol (Keller and Matile 1989; Moore et al. 1997) and these studies do not include measurements of phloem concentrations of mannitol.
Another sugar alcohol present in higher plants is sorbitol. Sorbitol is a major photoassimilate translocated in addition to sucrose in the phloem of woody Rosaceae, including all members of the economically important genera Malus (apple), Pyrus (pear) and Prunus (stone fruits such as peach, cherry, plum and apricot; Zimmermann and Ziegler 1975; Bieleski 1982; Moing et al. 1997). Sorbitol is synthesized in source organs from glucose-6-phosphate by the activity of aldose-6-P-reductase (Negm and Loescher 1981) converting glucose-6-phosphate to sorbitol-6-phosphate. Sorbitol-6-phosphate is further converted to sorbitol by a specific phosphatase. Sinks have little or no capacity to synthesize sugar alcohols like mannitol or sorbitol (Loescher and Everard 1996). To our knowledge, there is only one report on the intracellular distribution of sorbitol in leaf cells (Moore et al. 1997) and with the exception of peach (Moing et al. 1997) there are no data on polyol concentrations in the phloem sap of sugar–alcohol-translocating species.
In addition to mannitol and sorbitol, most plant species also contain the cyclitol myo-inositol (Zimmermann and Ziegler 1975) which is also produced from glucose-6-phosphate (Loewus and Loewus 1983). Myo-inositol and its derivatives are involved in membrane formation, cell wall synthesis, seed germination, and hormone responses as well as stress responses (Loewus and Loewus 1983). As an integral part of galactinol, myo-inositol also acts as a carrier of activated galactose that is transferred to sucrose, yielding raffinose and its polymeric derivates of higher order.
Knowledge of concentrations of sugar alcohols and sugars in subcellular compartments of leaf cells and in the phloem is important to understand possible modes of phloem loading. Therefore, we examined the subcellular concentrations of sorbitol, mannitol, myo-inositol, hexoses and sucrose in peach (P. persica, Rosaceae), celery (A. graveolens, Apiaceae), common plantain (P. major, Plantaginaceae), and sea plantain (P. maritima, Plantaginaceae) as well as the corresponding concentrations in the apoplast and in the phloem sap. Celery and common plantain were selected because much of what is known about the transport of mannitol and sorbitol has come from studies of these plant species, and more recently, the relevant transporters for sucrose as well as for sugar alcohols were cloned from these species. In addition, sea plantain and peach were selected because both species also translocate sorbitol but the former belongs to the group of halophytes and the latter to the group of woody plants. In this manner, different groups of sorbitol-translocating plants are included in the comparison. The sugar alcohols and sugars listed above are the main soluble carbohydrates in these plants. Based on the subcellular distribution, we calculated the concentration ratios of soluble carbohydrates between the different subcellular compartments as well as in compartments which are involved in phloem loading. In combination with results from morphological studies and data about sucrose and polyol transporters available in the literature the results are compared to predictions based on models of symplastic and apoplastic phloem loading that are currently discussed in the field.
Material and methods
Materials
Plants of A. graveolens var dulce cv Vert d´Elne (celery; supplied by R. Lemoine, University of Poitiers cedex, France), P. major (common plantain; supplied by N. Sauer, University of Erlangen, Germany) and P. maritima (sea plantain; supplied by N. Sauer, University of Erlangen, Germany) were grown in 2-l pots in compost soil in a green-house with supplemental illumination at a 15 h photoperiod and a 23/18°C day–night thermoperiod. Two-month-old plants were taken for the experiments. P. persica (peach), cv. Red Haven (Jenssen, Göttingen, Germany) was grown in 5-l pots in compost soil in a green house. Three-year-old plants were used for the experiments. Leaf samples were harvested in May and June.
Non-aqueous fractionation of leaf tissue
Leaves were cut from the plants after 9 h of the light period. The middle rib and larger veins were removed, and the samples were ground to a fine powder in liquid nitrogen in a precooled mortar. The leaf tissue was lyophilized at −25°C. The dry leaf powder was suspended in 20 ml of heptane:tetrachloroethylene mixture (density 1.27 g ml−1, the solvents were dried and stored over molecule sieve beads from Merck, Darmstadt, Germany) and ultrasonicated with 5-s pulses and 5-s breaks for a total sonication period of 95 s (Cell Disrupter B15, Bronson Sonifer). The suspension was then poured through a polyester monolen sieve with a pore size <40 μm, diluted 3-fold with heptane, and centrifuged for 10 min at 2,200g (Centrifuge 5810R, Eppendorf). The clear supernatant was discarded and the sediment resuspended in 3 ml of a heptane:tetrachloroethylene mixture (density 1.27 g ml−1). Two 200 μl aliquots were withdrawn for the determination of enzyme activity and carbohydrates in the unfractionated material. Two milliliter of the remaining material were added to an exponential heptane-tetrachlorethylene gradient with a density difference between 1.27 and 1.48 g ml−1 for common plantain and sea plantain, and between 1.27 and 1.45 g ml−1 for peach and celery. The bottom of the centrifugation tube contained a 2 ml cushion of a heptane:tetrachlrethylene mixture with the highest density (1.58 g ml−1). After centrifugation (25,000g, 2.5 h) six or seven fractions were collected from the centrifuge tube content. Each of these fractions was divided into two portions, one for assay of marker enzymes and the other for assay of carbohydrates. NADP-glycerine aldehyde phosphatase, PEP-carboxylase and α-mannosidase were taken as marker enzymes for stroma, cytosol and vacuole. Chloroplastic material (including stroma) appeared in the lower region of the gradient, the cytosolic compartment was found to be enriched in the middle region of the gradient, whereas vacuolar material was mainly found in the fraction of highest density. Chloroform methanol extracts were prepared from the aliquots for the determination of the carbohydrate concentrations (see next section: “Extraction of sugars and sugar alcohols from non-aqueous fractions”).
For the evaluation of the subcellular distribution of sugars and sugar alcohols between the stromal, cytosolic, and vacuolar compartment, a calculation procedure according to Riens et al. (1991) was used. This calculation method is based on the assumption that the metabolites are confined to the three compartments as indicated by the corresponding marker enzymes. The evaluation is done by a computer program testing all possible cases for the distribution of a certain metabolite between the three compartments, using increments of 1%; for example: (1) vacuole 100%, stroma 0%, and cytosol 0%; (2) vacuole 99%, stroma 1%, and cytosol 0%; (3) vacuole 99%, stroma 0%, and cytosol 1%. There are 5,151 possibilities for the distribution of a metabolite between the three compartments and the program uses possibility yields to calculate the best fit (agreement) with the experimental results. To avoid the results being falsified by analytical errors, the calculations are based on mean values obtained from measurements of at least five independent density gradient fractionations.
Extraction of sugars and sugar alcohols from non-aqueous fractions
Chloroform:methanol extracts were prepared to extract sugars and sugar alcohols from samples after non-aqueous fraction. The aliquots (see above) were dried and 5 ml chloroform:methanol (3:7, v/v) was added to the powder. The samples were homogenized until completely thawed and kept on ice for 30 min. The homogenate was then extracted twice with 3 ml water. The aqueous phases were combined and evaporated in a rotary evaporator. The dried residue was dissolved in 0.7 ml ultrapure water (Millipore), syringe-filtrated (0.45 μm cellulose-acetate; Schleicher and Schuell, Dassel, Germany) and stored at −80°C.
Collection of sieve tube sap
Sieve tube sap was obtained from severed stylets of the green-peach aphid, Myzus persicae (Sulz.). About 10 aphids were caged for about 5 h on the leaf. Their stylets were cut by a laser beam (Lohaus et al. 1995). The exuding phloem sap (10–500 nl) was collected in micro-capillaries (total volume 0.5 μl) and the volume of the exudate was determined by measuring the length occupied by the solution. Evaporation of the phloem sap was prevented by bringing the front edge of the capillary in close contact with the leaf surface and surrounding the end with a plastic cap. The humidity around the capillary was about 80%. Under these conditions no evaporation from reference capillaries was detectable. The samples were ejected into 50 μl of distilled sterile water and stored at −80°C.
Extraction of apoplastic washing fluids from leaves
Leaves were cut 7–8 h after the onset of illumination and carefully washed with deionized water. Leaves were placed into a syringe filled with 40 ml deionized water and infiltrated by pulling the plunger, producing a reduced pressure of approximately 20 kPa. Thereafter, intact leaves were blotted dry, positioned with the xylem wound up in a 10 ml vessel and centrifuged immediately at 200g, for 4 min at 4°C.
Due to the fact that infiltration of the apoplastic air space leads to a dilution of the apoplastic fluid, the solute concentrations in the apoplastic washing fluid were corrected by the ratio volume of the infiltration solution (which corresponds to the volume of the apoplastic gas space Vgas) to the volume of the apoplastic water space (Vwater). The ion concentration in the apoplast was calculated by multiplying the ion concentration in the apoplastic washing fluid by the dilution factor (=(Vwater + Vgas )/Vwater). The mean dilution factor of several C3 herbal dicot plant species is about six (Lohaus et al. 2001). Therefore, the concentrations of the apoplastic fluid were diluted about 6-fold by the infiltration procedure in common plantain, sea plantain and celery. The dilution factor in peach was only 2.7 because the apoplastic water space was 17%, expressed as percentage of total leaf water content (Moing et al. 1997) which corresponds to about 113 μl g−1 leaf water space (Table 3), and the apoplastic gas space was 190 μl (g FW)−1 (Table 3).
Cellular contamination of apoplastic washing fluids was quantified by comparing the activity of malate dehydrogenase in the apoplastic wash fluid with that in leaf extracts (Lohaus et al. 2001). According to this test, cellular cross-contamination was always below 0.1%.
Metabolite analysis
Sugars and sugar alcohols in the samples after non-aqueous fractionation, apoplastic washing fluids, and phloem sap were analyzed by HPLC. An ion exchange column (CarboMA10; Dionex Corp, Sunnyvale, CA, USA) was eluted isocratically with 600 mM NaOH (0.4 ml min−1) buffer for 60 min. Sugars were detected by a pulse amperometric detector with a gold electrode (ESA, Model 5200, Coulochem II, Bedford, MA, USA). Pulse setting was at 50, 700 and −800 mV for 500, 540 and 540 ms, respectively. Sugar standards were measured daily, and plant samples were diluted sufficiently to provide signals within the linear range of the detector response (50–500 μM). The metabolite amount was calculated from the peak area using Peaknet 5.1 software (Dionex, Idstein, Germany).
Electron microscopy and determination of subcellular volumes
Source leaves were cut into pieces of approximately 2 mm2 and prefixed in 5% glutaraldehyde (v/v), 5 CaCl2, and 50 mM sodium-cacodylate buffer, pH 7.0. After 3 h at 4°C, the fixed slices were washed four times for 15 min each in cold 50 mM sodium-cacodylate buffer, pH 7.0, fixed in 2% (w/v) osmium tetraoxide in cacodylate buffer and 0.8% (w/v) K4Fe(CN)6 for 3 h at 4°C. The samples were washed five times in distilled water and stained with 5% uranyl acetate. Samples were dehydrated by subsequent incubation in higher concentrations of acetone [30, 50, 70, 90 and 100% (w/v)]. The samples were then incubated in an acetone/epoxyresin mix (Spurr 1969) with increasing concentrations of epoxyresin [33, 66 and 100% (w/v)] for 1 h per concentration, and overnight and 4 h at 100% (w/v). Sample blocks were than trimmed with a razor and sectioned in an ultra-microtome (Leica Microsystems, Wetzlar, Germany) with a diamond knife. Slices of approximately 60 nm were placed onto copper grids (Plano, Marburg, Germany), stained with uranylacetate and 0.3% lead citrate in NaOH and observed in a transmission electron microscope (EM 400, Philips, Eindhoven, NL).
Electron micrographs of these sections were used for the evaluation of subcellular volumes according the principle of Delesse (1847): “the areal density of profiles on sections is an unbiased estimate of the volume density of structures” (Weibel and Bolender 1973; Winter et al. 1993; Farré et al. 2001). About 50 electron micrographs were taken from 2 independent fixation procedures and from each 6 different leaves. Low magnification and high magnification (>x1,000) were used to determine the areas of most compartments. The cross-sectional areas of subcellular compartments were quantified using analysis software (NIH image, public domain software, developed at US National Institutes of Health, available at http://rsb.info.nih.gov/nih-image/).
The relative volumes (percent of total) were converted to absolute volumes per unit mass by taking into account the water content per mass fresh weight.
Results
Subcellular distributions of sugars and sugar alcohols in leaf cells
Subcellular distributions of sugar alcohols, mono- and di-saccharides were measured by non-aqueous fractionation and HPLC in common plantain, sea plantain, peach and celery. The chosen method is based on the comparison of metabolite and marker enzyme distributions using a three-compartment calculation program (Riens et al. 1991). The method gives highly reproducible results for metabolites which are exclusively located (e.g., hexoses that are predominantly located in the vacuole). A higher variation is found for metabolites located in more than one compartment. The variability is greatest when the proportion found in a particular compartment is low. Farré et al. (2001) estimated that the limit of detection of a compound in a particular compartment is around 5% of the total amount in the tissue.
HPLC analysis of gradient fractions of common plantain, sea plantain, and peach showed that sucrose, hexoses (glucose and fructose), myo-inositol, and sorbitol were present in leaf cells in these three plants (Fig. 1a–c). Celery contains mannitol instead of sorbitol (Fig. 1d). In all four species the subcellular distribution of sorbitol and mannitol, respectively, was very different from those of hexoses and sucrose (Fig. 1a–d). Sorbitol (in common plantain and peach) and mannitol (in celery) were located in all three compartments, but predominantly in the vacuole (48–73%), followed by the stroma (19–27%) and the cytosol (8–25%). The distribution in sea plantain was different, and sorbitol was found mainly in the vacuole (88%) with only small portions in the stroma and in the cytosol. In contrast, in all four plants glucose and fructose were confined entirely to vacuoles (96–98%), and in common plantain and sea plantain sucrose was mainly distributed among the vacuolar and the cytosolic compartments (Fig. 1a, b). In peach and celery, a greater proportion of sucrose was found in the vacuolar compartment and a smaller part in the cytosolic compartment (Fig. 1c, d). The cyclitol, myo-inositol, was mostly present in the stroma of common plantain and sea plantain. In peach and celery the proportion was high in the stroma as well as in the vacuole, while the proportion found in the cytosol was always the lowest (Fig. 1c, d).
Fig. 1Percentage distribution of sugars and sugar alcohols among the vacuolar, stromal and cytosolic compartments of leaf cells from a common plantain, b sea plantain, c peach, and d celery. Data represent mean values ± SD from five to six independent fractionations
Subcellular volumes
The further conversion of the percentage distribution of sugars and sugar alcohols into concentrations requires an estimation of the volumes of the subcellular compartments of mesophyll cells. Leaves contain different tissues but in dicots about 75% of the aqueous volume of the leaf is occupied by mesophyll cells (Winter et al. 1993, 1994; Leidreiter et al. 1995). Table 1 shows the relative proportions of the vacuolar, chloroplastic and cytoplastic (sum of cytosol, peroxisomes, mitochondria, and nucleus) compartments of TEM micrographs of mesophyll cells. In all plant species analyzed the most voluminous compartment was the vacuole, occupying 68–85% of the total cell, followed by the chloroplasts (10–21%) and the cytoplasm (5–12%). The proportion of the vacuole was lowest in the woody plant peach whereas in common plantain and sea plantain mesophyll cells were dominated by the vacuole (82–85%). Accordingly, the relative proportions of chloroplasts and cytoplasm were smallest in these plants. The relative proportion of the combined volumes of mitochondria, peroxisomes, and nuclei of the total cytoplasmic volume has been estimated to be about 20% (Winter et al. 1993, 1994) and the proportion of the stroma volume of the total chloroplast volume about 50% (Winter et al. 1993, 1994). From the relative proportions mentioned above, the data from Winter et al. (1993, 1994) and the average water contents of leaf cells (Table 2) the volumes of the vacuolar, stroma, and cytosolic compartments can be estimated (Table 2).
Table 1Relative volumes (%) of the subcellular compartments within the total volume of mesophyll cells from common plantain, sea plantain, peach and celeryVacuole %Chloroplast %Cytoplasm %Common plantain82 ± 512 ± 46 ± 1Sea plantain85 ± 510 ± 45 ± 2Peach68 ± 721 ± 611 ± 4Celery72 ± 516 ± 412 ± 3Data were obtained from TEM micrographes (n = 37–42). Cytoplasm is defined as cytosol, peroxisomes, nucleus and mitochondria. Mean values ± SDTable 2Dry weight (n = 8), water and gas space (n = 12) of leaves and the volumes of subcellulare compartments of mesophyll cells from common plantain, sea plantain, peach and celeryCommon plantainSea plantainPeachCeleryDry weight (mg g−1 FW)147 ± 9124 ± 15336 ± 30140 ± 8Water space (μl g−1 FW)853 ± 9876 ± 15664 ± 30860 ± 8Gas space (μl g−1 FW)226 ± 48222 ± 21190 ± 44213 ± 33Volume vacuole (μl g−1 FW)701743452860Volume stroma (μl g−1 FW)50437068Volume cytosol (μl g−1 FW)42385882Mean values ± SD
Subcellular sugar and sugar alcohol concentrations
From the data on leaf carbohydrate contents, the subcelluar volumes, and the distribution of metabolites between the subcellular compartments the corresponding metabolite concentrations in the subcellular compartments were calculated. Sorbitol or mannitol, respectively, made up more than twothird of the total leaf content of soluble carbohydrates in all the plants (Table 3). The concentrations of sorbitol and mannitol, respectively, were high in all compartments of peach and celery (Table 3). Most of the sorbitol and mannitol were located in the vacuole (Fig. 1a–d) but due to the smaller volume cytosolic concentrations were calculated to be similar to those in the vacuole, and the highest level was found in the stroma (Table 3). The sorbitol concentration in the cytosol of sea plantain was lower (13 mM) than in the vacuole (87 mM) whereas in common plantain the vacuole showed the lowest sorbitol concentration (15 mM).
Table 3Whole leaf contents as well as sugar and sugar alcohol concentrations in the vacuolar, stromal and cytosolic compartments of leaf cells from common plantain, sea plantain, peach, and celeryWhole leaf content μmol (g FW)−1Concentration (mM)VacuoleStroma CytosolCommon plantain Sorbitol22 ± 3.115 ± 2.3118 ± 34133 ± 48 myo-Inositol0.7 ± 0.20.1 ± 0.18.8 ± 1.36.1 ± 0.7 Glucose and fructose2.4 ± 1.13.2 ± 0.21.4 ± 2.00.6 ± 0.7 Sucrose1.2 ± 0.10.9 ± 0.20.1 ± 0.212 ± 2.5Sea plantain Sorbitol73 ± 9.187 ± 8.0201 ± 14013 ± 8.4 myo-Inositol0.8 ± 0.20.2 ± 0.115 ± 0.40 ± 0 Glucose and fructose11 ± 2.515 ± 0.50.2 ± 0.50 ± 0 Sucrose1.4 ± 0.20.8 ± 0.30.1 ± 0.120 ± 4.9Peach Sorbitol148 ± 0.1220 ± 15461 ± 165290 ± 62 myo-Inositol1.5 ± 0.11.5 ± 0.48.5 ± 3.64.1 ± 3.3 Glucose and fructose30 ± 6.165 ± 15.0 ± 5.05.0 ± 5.0 Sucrose38 ± 2.164 ± 4.539 ± 26106 ± 15Celery Mannitol108 ± 12127 ± 7.0294 ± 30100 ± 39 myo-Inositol1.4 ± 0.11.1 ± 0.39.0 ± 1.91.7 ± 0.9 Glucose and fructose17 ± 2.028 ± 0.32.5 ± 1.72.9 ± 1.2 Sucrose35 ± 4.345 ± 3.35.2 ± 3.286 ± 28Mean values ± SD
In each plant species sucrose was mainly concentrated in the cytosol, followed by the vacuole and was lowest in the stroma, however, concentrations varied widely between the species (cytosol 12–106 mM, vacuole 0.8–64 mM, and stroma 0.1–39 mM; Table 4). These differences between the plant species reflect the differences in the total leaf contents (Table 3). The sucrose content in both plantain species was much lower than in peach and celery.
Table 4Carbohydrate concentrations in the cytosol of mesophyll cells of leaves as well as in the apoplast and in the phloem sap from common plantain, sea plantain, peach and celeryConcentration (mM) RatioRatioCytosolApoplast Phloem sapPhl/apoPhl/cytCommon plantain Sorbitol133 ± 485.5 ± 1.2422 ± 129773.2 Sucrose12 ± 2.50.3 ± 0.1645 ± 2252,15040 Sorbitol/sucrose11180.7Sea plantain Sorbitol13 ± 8.47.9 ± 2.5315 ± 1054024 Sucrose20 ± 4.90.3 ± 0.1355 ± 1121,18318 Sorbitol/sucrose0.7260.9Peach Sorbitol290 ± 6224.4 ± 9.5582 ± 90242.0 Sucrose106 ± 161.2 ± 0.6207 ± 511732.0 Sorbitol/sucrose2.7202.8Celery Mannitol100 ± 396.7 ± 3.3732 ± 1311097.3 Sucrose86 ± 281.2 ± 0.9389 ± 843244.5 Mannitol/sucrose1.25.61.9Mean values of n = 3–9 independent measurements are shown. Mean values ± SD
Of the compartments analyzed the highest concentration of glucose and fructose was consistently found in the vacuole. As the evaluation of the subcellular fraction was performed in increments of 1%, glucose and fructose concentrations found in the cytosol or stroma represent an upper limit of 1% for these compounds.
The concentration of myo-inositol was highest in the stroma, with intermediate values in the cytosol and lowest in the vacuole (Table 3). Myo-inositol, sorbitol and mannitol, respectively, made up between 90 and 99% of the total soluble carbohydrates in the stroma of the plant species.
Apoplastic and phloem sap concentrations
Carbohydrates found in the phloem sap of the four plant species investigated were sucrose and mannitol or sorbitol (Table 4). Concentrations were high in all cases, ranging between 200 and 700 mM. In the two Plantaginaceae sucrose concentrations, measured directly, were slightly higher than sorbitol concentrations, whereas in peach and celery the sorbitol and mannitol concentrations, respectively, were 3- and 2-fold higher than the sucrose concentration. Therefore, the ratios of sugar alcohol to sucrose concentrations for the four plants differ between 0.7 in common plantain and 2.8 in peach. No or only traces of glucose and fructose were found in the phloem, and the concentration of myo-inositol was in the low millimolar range (data not shown).
Sorbitol and mannitol, respectively, dominated the apoplast of all four plants, while sucrose was present at concentrations at or below 1 mM (Table 4). Nevertheless, the polyol as well as the sucrose concentrations in the apoplast were much lower than in phloem sap (Table 4). The concentration differences between the apoplast and the phloem were about 100-fold for mannitol, between 20- and 80-fold for sorbitol and between 170- and 2,200-fold for sucrose.
Discussion
Despite the central importance of the compartmentation of sugar alcohols in plant cells in relation to phloem transport, up to now the sugar alcohol distribution in subcellular compartments has been the subject of very few studies. We have used non-aqueous fractionation in addition to HPLC to study subcellular sugar alcohol distributions in common plantain, sea plantain, peach, and celery which translocate large amounts of sorbitol or mannitol within the phloem. It should be pointed out that the non-aqueous fractionation technique was developed for determination of subcellular concentrations of metabolites that are exclusively located in the mesophyll (Gerhardt and Heldt 1984). For other metabolites such as sugars, which are also present in tissues other than the mesophyll, the method tends to somewhat overestimate concentrations in the mesophyll cell compartments, because the amounts of metabolites in other tissues are assumed to be negligible. However, Voitsekhovskaja et al. (2006) have shown that concentrations of hexoses and sucrose in mesophyll cells measured either by single-cell technique or by non-aqueous fractionation were similar. A possible explanation is that e.g., the dense phloem tissue remains intact and will be removed from the other tissue during fractionation process (either in the sieve or in the bottom of the gradient; see chapter “Non-aqueous fractionation of leaf tissue”).
Sorbitol and mannitol concentrations are high in stroma and cytosol despite of a large vacuolar polyol depot
The main proportion of sorbitol and mannitol was found in the vacuolar compartment (48–87%; Fig. 1a–d) whereas in most species analyzed the highest concentrations were found in the stroma (Table 3). High concentrations of polyols in the stroma require either polyol-uptake or polyol-synthesis inside this compartment. Unfortunately, little is known about the intracellular distribution of the enzymes involved in sorbitol synthesis. Experiments with apple (Yamaki 1981) support a predominantly chloroplastic location for sorbitol-6-phosphate dehydrogenase. On the other hand, the intracellular immunocytochemical localization data for mannose-6-phosphate reductase, the key enzyme for mannitol synthesis, show that this enzyme is primarily cytosolic (Loescher and Everard 1996). So far no polyol transporter has been demonstrated to be localized in the envelopes of chloroplasts. The function of high concentrations of polyols in the stroma could be e.g., for osmoregulation because the concentration of several other metabolites or ions in this compartment is much lower than in the cytosol and in the vacuole (Table 3; Winter et al. 1993; Lohaus et al. 1998; Voitsekhovskaja et al. 2006).
Of the plants analyzed which produce sorbitol, only in sea plantain was the sorbitol concentration in the vacuole of mesophyll cells higher by about 7-fold than that in the cytosol (Table 3). The only previous report about subcellular distribution of sorbitol was of snapdragon (Moore et al. 1997) where up to 100% of sorbitol was found in the vacuole. However, the total leaf content of sorbitol was much lower in snapdragon than in plants in our study. A higher sorbitol concentration in the vacuole than in the cytosol, as in sea plantain, demonstrates that there is an active transport of sorbitol into the vacuole, because most likely sorbitol is either produced in the cytosol or in other cell compartments. So far no sorbitol transporter has been demonstrated to be localized in the tonoplast of mesophyll cells.
The high sorbitol concentration in the vacuoles of sea plantain indicates that sorbitol may have more functions in that species than just serving as a transport-form for carbon. Sea plantain is a more salt tolerant plant species than common plantain (Ahmad et al. 1979). In the study presented here no salt stress was given but the plants have taken up sodium from the soil used for plant culture. As a result the sodium content in common plantain leaves was 4.3 ± 1.1 μmol (g FW)−1, whereas sea plantain contained 36 ± 6.5 μmol (g FW)−1 which corresponds to the higher sorbitol content in the leaves in sea plantain (Table 3). Stoop et al. (1996) have discussed that mannitol may accumulate in the vacuole of salt-tolerant plant species for redistribution to the cytosol in response to stress, and recently Pommerrenig et al. (2007) demonstrated increased sorbitol contents in leaf cells of common plantain in response to salt treatment.
In leaves of celery three quarters of mannitol was located in the vacuole (Fig. 1d). In A. barclaiana (Voitsekhovskaja et al. 2006), parsley and snapdragon (Moore et al. 1997) the vacuole, stroma and the cytosol contained each onethird of leaf mannitol, but the total leaf content of mannitol was much lower in these three plants than in celery. Probably the higher leaf content of mannitol requires the deposition of a greater portion of mannitol in the vacuole. Despite the high proportion of mannitol in the vacuole, cytosolic and stromal mannitol concentrations were calculated to be similar to be higher than those in the vacuole (Fig. 1d and Table 3). Mannitol is synthesized in the cytosol of leaf cells (Rumpho et al. 1983) and similar concentrations of mannitol in the vacuole and the cytosol indicate that diffusion of mannitol between both compartments could be responsible for the pattern observed. On the other hand, mannitol is transiently stored in the vacuoles of parenchyma cells of fleshy petioles (Greutert et al. 1998). Similarly, Keller and Matile (1989) estimated a 3-fold higher mannitol concentration in the vacuole than in the cytosol of petiole cells, suggesting that an active uptake system of mannitol is present in the tonoplast. The vacuolar mannitol concentration higher in storage petioles than in source leaves may be a reflection of the different functions of these two organs in celery.
A large pool of myo-inositol is located in the stroma
A great portion of myo-inositol is localized in the stroma (Fig. 1a–d) which corresponds to earlier findings (Voitsekhovskaja et al. 2006; Moore et al. 1997). The high proportion of myo-inositol in the stroma also resulted in the highest myo-inositol concentration in this compartment (8–15 mM, Table 3), compared to other compartments. This stromal pool probably originates from its synthesis by the stromal isoform of the myo-inositol synthesizing enzyme, myo-inositolphosphate synthase (Adhikari et al. 1987). In the halophyte, Mesembryanthemum crystallinum, myo-inositol synthesis is increased in response to salt stress (Ishitani et al. 1996). Leaves of sea plantain contained a 8-fold higher salt content than those of common plantain (see above). Despite this difference, the content of myo-inositol was very similar in both plants in contrast to the sorbitol content (Table 3). Therefore, it seems for plantain that the sorbitol concentration is correlated with the sodium concentration rather than the myo-inositol concentration, even under low sodium conditions.
The subcellular distribution of hexoses and sucrose is similar in plant leaves with and without sugar alcohols
Hexoses are often confined to the vacuole (Riens et al. 1991; Winter et al. 1992). Figure 1a–d shows that these findings are also true for the four species analyzed here: concentrations of glucose and fructose were always higher in the vacuole than in the cytosol or in the stroma (Table 3). Both facilitated diffusion and active transport have been described for vacuolar uptake of glucose. Facilitated diffusion has been discussed for the transport of glucose across the tonoplast in celery (Daie 1987). Heineke et al. (1994) suggested that leaf vacuoles may contain transporters for the active uptake of hexoses. However, Martinoia et al. (2000) noted that accumulation of hexoses within the vacuole is not a proof for energized hexose uptake, because the vacuole contains acid invertases, allowing for enzymatic production of hexoses on the vacuoles.
Between 50 and 80% of the sucrose detected in the four species analyzed were located in the vacuole (Fig. 1a–d). However, due to the smaller volume of the cytosol relative to that of the vacuolar compartment, cytosolic sucrose concentrations were calculated to be similar to or even higher than those in the vacuole (Table 3). A similar distribution has also been reported for leaves of other species (Riens et al. 1991; Winter et al. 1992). The lowest vacuolar sucrose concentration was found in the two Plantaginaceae and may be related to the overall low sucrose content in the leaves of these two species (Table 3). Recently Endler et al. (2006) have localized sucrose transporters in the tonoplast of barley (HvSUT2) and Arabidopsis (AtSUT4) and proposed that sucrose transporters are involved in vacuolar transport.
Concentration ratios between the cytosol of mesophyll cells and the phloem were similar for sucrose and sugar alcohols
Sugar alcohols and sucrose are the major compounds translocated in the four species analyzed. The phloem concentrations of sorbitol and mannitol (Table 4) were in the same range as that of sucrose (300–700 versus 200–700 mM) although the ratio of sorbitol, or mannitol, to sucrose varied between 0.7 and 2.8. High concentrations of sugar alcohols in the phloem indicate that a high proportion of assimilated carbon is exported in this way. Moing et al. (1997) estimated that sorbitol accounts for 60–90% of the carbon exported from the leaf in peach and in celery, while mannitol was shown to account for 10–60% (Daie 1986).
In the four plants the sucrose concentration was always higher in the phloem sap than in the cytosol of mesophyll cells although the concentration ratio of sucrose between the cytosol of mesophyll cells and the phloem varied between 2- and 40-fold (Table 4). The different ratios arose from variable cytosolic sucrose concentrations rather than by different phloem concentrations in the four plants (Table 4). With the exception of common plantain the ratios of both, sorbitol and mannitol, between the phloem and the cytosol of mesophyll cells were similar to the corresponding ratios of sucrose. In common plantain the overall sucrose concentration was relatively low which resulted in a higher ratio for sucrose than for sorbitol.
Concentration ratios between the apoplast and the phloem for sucrose and sugar alcohols with relation to the phloem loading mode
Gamalei (1989) has shown that the members of Plantaginaceae display a virtually complete symplastic isolation of the sieve element-companion cell complex, as indicated by a very low plasmodesmal abundance between phloem companion cells and bundle-sheath cells. Sucrose and sorbitol were present in the apoplast of both Plantaginaceae, however, the concentrations were much lower than in phloem sap (Table 4). Therefore, the loading of both sucrose and sorbitol into the phloem must be energized. This is consistent with the observation that sucrose transporters (PmSUC1, PmSUC2, and PmSUC3; Gahrtz et al. 1994; Barth et al. 2003) as well as polyol transporters (PmPLT1 and PmPLT2; Ramsperger-Gleixner et al. 2004) were cloned from P. major. The function of the putative transporter proteins in phloem loading was corroborated by their specific localization in companion cells of source leaf phloem (PmSUC2, Stadler et al. 1995; PmPLT1 and PmPLT2 Ramsperger-Gleixner et al. 2004). The Km value of PmSUC2 for sucrose was about 1 mM (Gahrtz et al. 1994) and the Km value of PmPLT1 for sorbitol was about 12 mM (Ramsperger-Gleixner et al. 2004), which are in the same range as the apoplastic sucrose and sorbitol concentrations (Table 4). No data of transporters exists for sea plantain, but the concentration gradients for sucrose as well as those for sorbitol strongly suggest active transport systems for both carbohydrates. Together, the data support apoplastic transfer of sucrose and sorbitol in the members of Plantaginaceae.
Pathway and mechanism of phloem loading in peach are still a matter of debate (Moing et al. 1997). The phloem structures of Rosaceae trees were shown to be of the intermediate-type (moderate numbers of plasmodesmal connections between mesophyll and companion cells; Gamalei 1989). Therefore, the mechanism of phloem loading in Prunus species could be symplastic, apoplastic or mixed. In apoplastic loaders, sucrose or sorbitol destined for phloem transport has to be released from the mesophyll cells into the apoplast, whereas in symplastic loaders, sugars and sugar alcohols synthesized in the mesophyll and destined for the phloem transport, are expected to stay in the symplast. Thus, it could be expected that the percentage of these carbohydrates in the cytosol of mesophyll cells in putative symplastic loaders would be much higher than in apoplastic loaders. Sorbitol concentrations were high in the phloem sap of peach (Table 4, Moing et al. 1997) and in addition to sucrose sorbitol is in general a major photoassimilate translocated in the phloem of woody Rosaceae (Bieleski 1982). The concentration ratios for sucrose and sorbitol between the cytosol of mesophyll cells and the phloem were about 2-fold for each carbohydrate (Table 4). These are the lowest ratios among the species analyzed and much lower than those determined for apoplastic phloem loaders (ratio of sucrose 5–30; Lohaus et al. 1995; Lohaus and Möllers 2000). A 2-fold concentration ratio for sucrose corresponds to the gradient found in Alonsoa meridionalis, a Scrophulariaceae with a symplastic phloem loading mode (Knop et al. 2001). A lower concentration ratio is obviously easier to balance or even reverse, depending on the metabolic situation, than a steeper ratio. Therefore, the possibility of phloem loading of sucrose and sorbitol by simple diffusion cannot be ruled out. Turgeon and Medville (1998) were unable to detect accumulation of sucrose against the concentration gradient in minor veins of willow leaves, representing another woody species.
On the other hand, leaf infiltration with p-chloromercuribenzenesulfonic acid (PCMBS) was found to inhibit sugar phloem transport in peach and Moing et al. (1992) further concluded from this result that apoplastic phloem loading predominates in peach. Unfortunately up to now no sucrose or sorbitol transporters were cloned from peach as they had from other Rosaceae (Prunus cerasus, Gao et al. 2003; Malus domestica, Watari et al. 2004). Watari et al. (2004) reported the existence of three sorbitol transporters in apple source leaves with apparent Km values for sorbitol between 0.7 and 3.2 mM. The apoplastic sorbitol concentration in apple leaves was not detected but the corresponding concentration in peach was about 25-fold higher (Table 4).
Sucrose and sorbitol uptake in plasma membrane vesicles from peach leaves exhibited saturated kinetics (Marquat et al. 1997). These results suggest that uptake of sorbitol and sucrose is carrier mediated. Active absorption of sucrose was completely inhibited by PCMBS, contrary to the absorption of sorbitol (Marquat et al. 1997). The activities of sorbitol transporters from Rosaceae heterologously expressed in yeast were also not or only slightly inhibited by PCMBS (Gao et al. 2003; Watari et al. 2004). Ramsperger-Gleixner et al. (2004) have shown that sorbitol transport by PmPLT1, but not that driven by PmPLT2, is inhibited by PCMBS. Therefore, PCMBS sensitivity of sorbitol transport can vary and the insensitivity of transport processes against PCMBS conclusively does not rule out that carriers are involved.
Unfortunately, the type of phloem loading in peach is still an open question; the available data are consistent with either an apoplastic or a symplastic mode of loading or, more likely, a combination of both.
Celery is the only plant species analyzed here which translocates mannitol in addition to sucrose. The concentration ratio of sucrose between the apoplast and the phloem sap was about 300-fold and the ratio for mannitol was about 100-fold (Table 4). Studies of metabolite uptake into plasma membrane vesicles from phloem tissues of celery petioles by Salmon et al. (1995) showed mannitol- as well as sucrose-proton-co-transport, indicating that in this species mannitol and sucrose transporters may be involved in phloem loading. Noiraud et al. (2000) have cloned two sucrose transporters from celery (AgSUT1, AgSUT2). AgSUT1 is expressed in mature leaves and phloem of petioles. Noiraud et al. (2001) have also cloned a cDNA of a mannitol transporter (AgMaT1) of celery which is not sensitive to PCMBS. The expression profile for AgMaT1 in source leaves and phloem was in agreement with a role in phloem loading of mannitol in celery. The AgMaT1 protein expressed in yeast cells exhibited a Km value for mannitol uptake of about 0.3 mM (Noiraud et al. 2001). The apoplastic mannitol concentration was 25-fold higher (Table 4). In summary, celery shows all characteristics of a typical apoplastic phloem loader.
The comparison of common plantain, sea plantain, peach, and celery indicates that different modes of phloem loading are employed by different plant families, which may in part be related to the respective ecophysiological requirements.

phloem plantago prunus apium sorbitol mannitol


Complementary and Alternative Medicine for Chronic Prostatitis/Chronic Pelvic Pain Syndrome

To discuss challenges concerning treatment for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and review complementary and alternative medical (CAM) therapies being evaluated for this condition, we performed a comprehensive search of articles published from 1990–2005 using the PubMed, Medline databases. Data from the articles were abstracted and pooled by subject. Keywords cross-searched with CP/CPPS included: complementary, alternative, integrative, therapies, interventions, nutrition, antioxidants, herbs, supplements, biofeedback and acupuncture. Listed articles with no abstracts were not included. Various CAM therapies for CP/CPPS exist including biofeedback, acupuncture, hyperthermia and electrostimulation. Additionally, a variety of in vitro and in vivo studies testing herbal and nutritional supplements were found. Saw palmetto, cernilton and quercetin were the most frequently tested supplements for CP/CPPS. Although many CAM therapies demonstrate positive preliminary observations as prospective treatments for CP/CPPS, further exploratory studies including more randomized, controlled trials are necessary for significant validation as treatment options for this complex disorder.
Introduction and Characterization of Prostatitis
Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is of significant interest in urology and accounts for up to 2 million office visits per year (1). Currently, there are multiple approaches to the management of CP/CPPS depending on the classification of the related symptoms. However, there are no absolute findings or laboratory tests employed and diagnosis is often one of exclusion.
In 1995, the National Institutes of Health established an International Prostatitis Collaborative Network in order to construct a new classification of prostatitis syndromes and better define chronic prostatitis (2,3). The categories are now documented as follows:
Category 1. Acute bacterial prostatitisCategory 2. Chronic bacterial prostatitisCategory 3. Chronic prostatitis/chronic pelvic pain syndrome
A. InflammatoryB. Non-inflammatoryCategory 4. Asymptomatic inflammatory prostatitis.
While most patients report a primary subjective symptom of local pain and/or dysuria, the clinical presentation of acute versus chronic prostatitis varies. Laboratory cultures are standardly employed to detect bacterial involvement and this testing along with other diagnostic criteria determines each classification. Men with Category 1, acute prostatitis, frequently present with dysuria, fever, malaise, myalgia (non-specific) and positive culture analysis that often reveals coliform bacteria. This imparts to standard antibiotic treatment with good prognosis for recovery and minimal recurrence.
Patients with Category 2, chronic bacterial prostatitis, present with similar symptoms as those with acute prostatitis. However, the frequency of symptoms (duration >3 months), recurrent urinary tract infections and additional diagnostic tests including analysis of lower urinary tract cultures contribute to its diagnosis as Category 2 prostatitis (2).
Men with Category 4, asymptomatic inflammatory prostatitis, do not present with subjective symptoms. This diagnosis is often discovered via laboratory findings such as the positive presence of white blood cells in prostatic secretions or in prostate tissue during routine evaluation for other disorders (1).
Comparative to the total number of prostatitis cases reported, the majority of representative cases are Category 3, CP/CPPS (1,4). This diagnosis is usually one of exclusion, as bacterial etiology acute or chronic is ruled out. Other exclusion criteria include urogenital cancer, urethral stricture and neurologic disease affecting the bladder. However, the patient may still present with polyuria, dysuria, generalized myalgia or specific pelvic pain, urethral discharge, voiding dysfunction, sexual dysfunction and negative impact on quality-of-life (QOL). The presentation of this symptom set is now termed Category 3, CP/CPPS. Categories 3A and 3B are further differentiated by the presence or absence of inflammatory blood cells in prostatic secretions and seminal fluid, respectively (4,5). Table 1 further outlines the characteristics and treatment options of Category 3 prostatitis.
Due to the complexity in diagnosing CP/CPPS, the National Institutes of Diabetes and Digestive and Kidney Diseases funded the Chronic Prostatitis Collaborative Research Network (CPCRN) in 1995 (2,3). This network was fundamental in the construction and validation of the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI), which was implemented in 1999 (3). The index has become a valid measure that quantifies the qualitative experience of men with CP/CPPS and addresses three different aspects of CP/CPPS: pain, function and QOL (3,6).
The formation of the CPCRN and the advent of the NIH-CPSI have better characterized diagnosis and treatment for CP/CPPS, but challenges still exist. While standard treatment options including anti-inflammatory agents, analgesics and alpha-blockers are often prescribed, impact on QOL is another factor often overlooked in the treatment and management of CP/CPPS (1,4,5,7). The focus on QOL, anecdotal data, epidemiological studies and the increased popularity and validation of herbal, complementary and alternative medicine (CAM) has led to exploration of the utility of CAM therapies as treatments for CP/CPPS (8). CAM therapies including biofeedback, acupuncture, heat therapy, electrostimulation, herbal and nutritional supplements will be discussed below.
CAM Background
CAM, as defined by the National Center for Complementary and Alternative Medicine (NCCAM), is a group of diverse medical and health care systems, practices and products that are not presently considered part of conventional medicine. Conventional medicine is further defined as medicine as practiced by holders of MD (medical doctor) or DO (doctor of osteopathy) degrees (9). Though the list of what therapies or practices considered to be CAM changes continually, the pool of both practitioners of CAM modalities and patients utilizing CAM services continues to grow within the United States and globally (9).
The inclusion of CAM practices in urology is also being implemented in the clinic. Many groups such as the Committee of Complementary and Alternative Medicine within the American Urological Association (AUA) recognize the integration of non-conventional therapies into urological clinical practice. Additionally, both public demand for CAM therapies and their testing and validation within health science research centers has increased (8,10,11). It has been suggested that many urological conditions possessing subjective and QOL components such as in benign prostatic hyperplasia (BPH), chronic prostatitis, voiding, erectile dysfunction and cancer prevention and survivorship might be particularly amenable to CAM treatment strategies (10). This review will focus on current CAM therapies found in the literature for CP/CPPS.
CAM Therapies for CP/CPPS
Biofeedback
Biofeedback therapy is considered a mind–body technique that utilizes a monitoring machine to assist people in controlling bodily functions such as heart rate, blood pressure and muscle tension. This therapy has been studied for its efficacy in urological conditions such as incontinence, prolapse, pediatric voiding dysfunction and CP/CPPS (12–17). The hypothesis of biofeedback's mechanism of action in treating CP/CPPS is based on the principle that maximum muscle contraction prompts maximum muscle relaxation. This mechanism addresses the chronic pain aspect of CP/CPPS and focuses on muscular reeducation, which may ultimately provide symptom relief (13–15).
Two studies testing the value of biofeedback therapy for CP/CPPS yielded positive results. The first study assessed 62 patients who were refractory to conventional therapy (such as antibiotics and/or alpha-blockers) for greater than half a year. These patients were treated utilizing the Urostym Biofeedback equipment five times a week for 2 weeks with a stimulus intensity of 15–23 mA and duration of 20 min. The NIH-CPSI index noted a significant overall reduction in score (P < 0.01) and no side effects were reported during the trial (13).
A second pilot study evaluated biofeedback therapy in 19 men with pelvic floor tension and CP/CPPS. These results demonstrated significant improvement in pain scores as measured by the AUA symptom index (P = 0.001). While this study focused on testing the effect of biofeedback therapy in treating the symptoms associated with CP/CPPS, it also implicated the presence of pelvic floor tension contributing to pain and the paramount importance of muscular reeducation for its treatment (15). These initial, positive biofeedback studies may warrant larger randomized clinical trials to confirm safety and efficacy as well as explore the mechanism of action of biofeedback therapy.
Acupuncture
Acupuncture is a traditional Chinese method of medical treatment involving the insertion of fine, single-use, sterile needles in acupoints according to a system of channels and meridians that was developed by early practitioners of Traditional Chinese Medicine (TCM) over 2000 years ago. The needles are stimulated by manual manipulation, electrical stimulation or heat (18). Currently, acupuncture is often used with TCM and it is a recognized health profession with strict licensure and regulatory status in 40 states (19). Common applications include acupuncture as a complementary therapy for cancer patients undergoing chemotherapy or radiotherapy, for conditions involving pain such as migraines and back pain, and for relieving the impact of stress among patients with chronic conditions. The precise physiological mechanism of action of acupuncture is unknown but a variety of hypotheses exist. For example, acupuncture analgesia is thought to be mediated by central nervous system (CNS) mechanisms of pain control via the release of specific neurotransmitters, such as endorphins (20–23). Additionally, there are significant data which suggest that acupuncture treatment can decrease inflammation and relieve pain (24,25).
Data suggesting the ability of acupuncture treatment to decrease pain, positively impact QOL and potentially modulate inflammation and/or affect the CNS has suggested it as potential therapeutic option for men with CP/CPPS. While a number of studies listed in other journals test the utility of acupuncture treatment for CP/CPPS (26) only two medline listed pilot studies are shown testing acupuncture treatment in patients with CP/CPPS.
The first study examined whether acupuncture improved pain, voiding symptoms and QOL in 12 men with CP/CPPS. This study reported a significant decrease in total NIH-CPSI pain, urinary and QOL scores (P < 0.05) over 6 weeks of treatment and an average 33 weeks of follow-up (27). The mechanism of action addressed in this paper suggests a neuropathic model of CP/CPPS and the hypothesis that acupuncture, if considered a neuromodulatory therapy, may provide a therapeutic option for men with CP/CPPS (27).
A second study tested acupuncture treatment for CP/CPPS patients with intrapelvic venous congestion. This study of 10 patients receiving 5 weeks of acupuncture treatment also reported a significant decease in NIH-CPSI pain and QOL scores (P < 0.05, P < 0.01). While the study reported no serious adverse events, the mechanism of action was not addressed (28). The promising clinical outcome of both studies testing acupuncture for CP/CPPS implies that larger studies are required to confirm the utility of acupuncture in this patient population.
High Frequency Electrostimulation
Only one study in English was found utilizing electrostimulation for chronic prostatitis. This study tested a new, high frequency, urethral–anal prototype stimulation device in men with CP/CPPS twice weekly for 5 weeks. The results demonstrated a significant decrease in the NIH-CPSI (P = 0.0002) with no urethral, anal complaints or other side effects (29). The authors suggest that due to the positive results, simple technology and ability to be self-administered, this new device may have utility in the treatment of CP/CPPS. However, further studies and standardization of the electrostimulation device are essential.
Hyperthermia
Anecdotal evidence and a few clinical trials have suggested heat therapy or hyperthermia as a treatment option for men with CP/CPPS. Its mechanism of action is based on the application of heat to the prostate to relieve pain. An excellent review by Zeitlin (30) discusses the lack of literature concerning hyperthermia and CP/CPPS. The review notes a variety of pitfalls in hyperthermia research including variation and lack of standardization of treatment. These concerns are applicable to both the type of heat utilized such as interstitial heat or microwaves and variation in its application, either transrectally or transurethrally. The review also suggests that the hyperthermia instruments used were not validated and outcome measures were subjective. However, the review implies that utilization of a quantitative assessment tool, applied statistics and greater documentation of therapy type may offer hyperthermia a better opportunity to be evaluated as a potential therapy for CP/CPPS (30).
We also discovered the paucity of literature described by Zeitlin and only three listed clinical trials utilizing hyperthermia. The first study analyzed a group of 45 men with chronic abacterial prostatitis or prostadynia who underwent 6 weekly, 1 h sessions of local deep microwave hyperthermia (42.5 +/−0.5°C) to the prostate. Although the authors report encouraging results in the decrease of pain, these subjective patient assessments were not quantified by the NIH-CPSI or other index for CP/CPPS (31).
A second abstract discussed a randomized, sham-controlled comparative study utilizing transrectal microwave hyperthermia in 80 men with CP/CPPS. While this abstract noted a 75% symptomatic improvement in the treatment group, the study was available as an abstract only with no statistical significance or descriptive methodology reported (32). A third study also tested transrectal microwave hyperthermia for both chronic non-infectious and infectious prostatitis. While the study design incorporated obtaining measurement of prostatic secretions, uroflowmetry and transrectal color Dopplerographic mapping, the results from the study were not abstracted as the article was in Russian (33).
Herbal and Nutritional Supplements
Herbal and nutritional supplement therapies have been most widely investigated for their utility in CP/CPPS and other prostate conditions such as BPH (34–40). While many formulations have been cited for their use in a wide variety of urological conditions, saw palmetto, pollen extract and quercetin were the supplements found with specific application to CP/CPPS. While most herbal and nutritional supplements contain a wide variety of synergistic ingredients upon compositional analysis, some of the active components such as phytosterols or antioxidants are listed in Fig. 1.
Saw palmetto
Saw palmetto (Serenoa repens) garnered much attention in urology based on a great deal of anecdotal evidence regarding its prostate specific properties. It is widely used in many Asian, African and European countries and compositional analysis of the berry of S. repens exhibits sterols and free fatty acids as its major constituents (41). Initial studies suggested that the efficacy of saw palmetto may be similar to that of the pharmaceutical enzyme inhibitors such as finasteride. This preliminary data prompted the exploration of mechanism, utility and efficacy of saw palmetto in in vitro analysis and in clinical trial settings. While a number of trials have examined saw palmetto use for symptoms related to BPH (34–39), only a few have focused on it specifically for CP/CPPS (42–44).
The first study compared the safety and efficacy of saw palmetto berry supplement versus finasteride in men with Category 3, CP/CPPS. This prospective, open label 1 year study randomized 64 men to the saw palmetto or finasteride group, respectively. After 1 year of treatment, the NIH-CPSI score decreased from 23.9 to 18.1 in the finasteride group (P < 0.003) and from 24.7 to 24.6 in the saw palmetto group (P = 0.41). While significance was only achieved in the finasteride treatment arm, it was notable that at the end of the trial 41 and 66% of participants opted to continue the therapies of saw palmetto and finasteride, respectively, regardless of achieved statistical significance (42).
The second clinical trial from China examined the effects of prostadyn sabale capsules containing saw palmetto berry in patients with CP/CPPS. While 125 men reported positive outcome and the NIH-CPSI was used as a primary end point, the article is only available in Chinese and the manufacturer, active constituents of the capsules and statistical significance was not reported (43).
A third multicenter study testing a saw palmetto abstract called Permixon analyzed the response of Permixon therapy in 61 patients with Category 3B prostatitis. While 65% of the Permixon group reported improvement based on the Patients Subjective Global Assessment (SGA), the total NIH-CPSI and the pain, voiding and QOL/impact domains of the NIH-CPSI, statistical significance was not reported (44). Additionally, prostate volume was unchanged in both the treatment and control groups. While this multicenter study suggests that Permixon may provide clinical benefit for CP/CPPS 3B, the dosages and components of the Permixon product were not listed in the abstract.
The assessments of saw palmetto studies for CP/CPPS are far fewer than those for BPH. However, marked and continued progress in molecular studies, increased mechanistic data and more clinical trials in CP/CPPS are warranted to ascertain the utility and reproducibility of saw palmetto use in men with chronic prostatitis.
Pollen Extract: Cernilton
Pollen extract is traditionally collected from the flowers of various plant types and it contains carbohydrates, fat, protein, vitamins and minerals (41). The particular pollen extract named cernilton has been suggested to benefit a variety of urological conditions. Anecdotal evidence and references from traditional herbal texts have implicated cernilton's potent anti-inflammatory properties and potential in treating symptomatic relief of urinary pain and dysfunction often present in both CP/CPPS (41) and BPH (45,46). In vitro studies demonstrate a variety of experimentation on this particular extract including histopathological analysis of its effect on cell proliferation, apoptosis, serum cytokines and testosterone (47,48). The literature also lists quite a few clinical trials on pollen extract; however, five are in the Japanese language and one in the German language (45,48–52). While many of these studies report the positive activity of pollen extract and suggest its usefulness for CP/CPPS, data from these studies were not abstracted due to unavailability and translation of the articles.
One available study testing pollen extract reported a 78% favorable response of men with chronic prostatitis taking Cernilton® pollen extract at a dosage of 1 tablet TID for 6 months (53). While this study reported favorable results, the study was published in 1993 and similar subsequent larger phase clinical trials are not evident to further elucidate the possible utility of pollen extract in men with CP/CPPS.
Quercetin
Quercetin is known chemically as a mixture of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and 3,3′,4′,5,7-pentahydroxy flavone. It belongs to a group of polyphenolic substances known as flavonoids and is a member of the class of flavonoids called flavonols. It is commonly found in the plant kingdom in the rinds and barks of certain foods such as onions, grapes and green tea (41). Since quercetin is thought to have antioxidant, anti-inflammatory, antiviral, immunomodulatory, anticancer, gastroprotective and antiallergy activities it has been studied for a variety of conditions (54–56).
One prospective, randomized, double-blind, placebo-controlled trial was performed to test the action of this bioflavonoid in men with CP/CPPS. This placebo-based study assessed 30 men with CP/CPPS to receive the bioflavonoid quercetin, 500 mg twice daily or the placebo pill for 1 month. Significant change in the NIH-CPSI score was observed in the quercetin (P = 0.003) versus the placebo group, who had an insignificant mean improvement in the NIH-CPSI score (57). While this was the only clinical trial found testing quercetin for CP/CPPS, the positive outcome supports the need for further study including cost analysis of quercitin therapy in this patient population.
Other Herbal Agents for Prostatitis
Finally, a wide variety of herbal compounds not previously discussed were found during this review, many of which are commonly used in the TCM herbal material medica. Some of the compounds are Nan mi qing capsules containing Rheum palmatum and Rx. astragalus memberanaceus, Ye Ju Hua Shuan, an herbal suppository of Flos Chrysanthemi Indici and a variety of other formulae or capsules of which the ingredients were not listed (58–62).
Initial in vitro studies examining some of the herbal compounds effects on markers of inflammation such as thromboxanes (TBX2) and 6-keto-PGF1-α are promising as the link between chronic inflammation in CP/CPPS is still under investigation (58). While the preliminary reports of additional herbal compounds are encouraging, a number of difficulties exist in the extraction of this data. Namely, the articles are in the Chinese or other language and data including the ingredients, constituents and other practices including good manufacturing are unable to be assessed. While this conundrum exists for many herbal agents despite their historical use in a variety of traditional herbal medicine systems, testing of safety and utility are still necessary. However, the variety of herbal agents available offers a large source to draw from and the possibility that other herbal products might be beneficial in the treatment of CP/CPPS.
Conclusions
A summary of the reviewed literature is outlined in Table 2 and a variety of CAM modalities tested in patients with CP/CPPS include practitioner-based therapies such as biofeedback, hyperthermia, acupuncture and electrostimulation, and biological-based therapies including herbal and nutritional supplements. As the challenges in treating this complex and chronic disorder remain, further evidence of efficacious CAM treatment options for men with CP/CPPS is needed. Thus far, promising data on the function and efficacy of certain CAM therapies suggest their potential as treatment options for this patient population. Further exploratory studies including more in vitro studies of herbal products, mechanistic data, cost analysis and randomized, controlled trials will assist in validation of certain CAM therapies as permanent treatment options for men with CP/CPPS (63,64).

alternative medicine
chronic prostatitis
herbs
supplements
biofeedback
acupuncture
hyperthermia
complementary medicine