the relationship between the relative breast cancer ( bc ) risk and seaweed intake among humans is only now unfolding . a small body of research , both in vivo and in vitro , suggests seaweed may be useful in bc prevention ( funahashi et al . seaweeds are specifically used to treat tumors in traditional chinese medicine and japanese folk medicine . on a population level , those people for whom seaweed is a regular part of their diet , most notably in japan , have dramatically lower rates of bc ( hebert and rosen 1996 ; kodama et al . epidemiologic studies done in japan in the 1980s , before westernized diets were common , reported that japanese women had one out of three the rate of premenopausal bc and one out of nine the rate of postmenopausal bc ( reddy et al . bc incidence rates for women in japan are 20/100,000 compared with the us average of 118/100,000 ( us cancer statistics working group 2007 ) . although genetic predisposition has been proposed , when rates among migrants from japan to the usa are compared , bc incidence almost doubles after 10  years of residence in the usa ( 20/100,000 to 35/100,000 ) ( shimizu et al . american women who develop bc have significantly better survival rates than other american ethnic groups ( kanemori and prygrocki 2005 ) . on the other hand , asian  american women over 50  years of age living in los angeles , especially japanese  american women , have one of the most rapidly increasing bc incidence rates ( deapen et al . these data support the hypothesis that lifestyle changes and possibly gene  nutrient interactions are important in bc susceptibility . seaweed is a typical part of east - asian diets , although consumption varies widely among individuals ( fukuda et al . brown seaweeds have no land equivalents in terms of their specific components of fiber ( alginate ) , primary carotenoid ( fucoxanthin ) , sulfated polysaccharide ( fucoidan and laminarin ) , and polyphenol defense compounds , each of which has been reported to have strong anti - cancer activity ( kang et al . 2012 ; kotake - nara et al . 2005 ; koyanagi et al . 2003 ; miao et al . many in vivo and in vitro studies of dietary seaweed report decreased angiogenesis and increased apoptosis of tumor cells ( konishi et al . 2005 ) , inhibition of tumor cell adhesion and metastasis ( liu et al . 2005 ) , and enhanced immune responses ( maruyama et al . ( 2000 ) have investigated seaweed activation of the urokinase plasminogen system pathway , urokinase - type plasminogen activator receptor ( upar ) was not specifically studied . based on the wide range of antitumor effects , we investigated the possibility that seaweed could affect upar concentrations in women who consume seaweed . the upar ( cd87 ) , is the membrane receptor for serine protease and is central to plasminogen activation and pericellular proteolysis . upar is an extracellular receptor involved in multiple signaling pathways important in maintaining homeostasis , directly interacting with 42 proteins , affecting the extracellular matrix ( ecm ) , cell adhesion , inflammation , immune function ( complement ) activation , blood coagulation , and tissue repair ( eden et al . 2011 ) . increased concentrations have been shown to be associated with the presence of cancer , and increased cell surface shedding of upar is characteristic of more rapid cancer progression ( boonstra et al . we therefore included evaluation of upar as a possible biomarker for seaweed activity that might be related to bc prevention . to further assess whether a dietary seaweed intervention could alter protein expression in urine and serum in a non - seaweed - consuming population of healthy postmenopausal women , we used surface - enhanced laser desorption / ionization  time of flight coupled with mass spectrometry ( seldi - tof - ms ) . proteomic analyses have been used to identify cancer biomarkers with high sensitivity and specificity , including those related to bc ( gast et al . seldi has also been shown to be sensitive enough to be used to identify changes in serum associated with the addition of a novel food ( green tea ) ( tsuneki et al . the human subjects research review board of the us army medical research and materiel command , the palmetto hospital health alliance ( columbia , sc ) , and the university of south carolina institutional review boards approved the study ( irb number , 99 - 104 ) . inclusion criteria were : being healthy , postmenopausal ( verified by follicle stimulating hormone ( fsh ; 23.0116  miu ml ) ) , no allergies to seaweed , soy , shellfish or iodine ; no current use of tobacco ; no hormone replacement therapy or , for bc survivors , no chemotherapy or radiation treatments within the preceding 6  months and no history of cancer ( other than bc or squamous cell skin cancer ) within the previous 20  years ; no current gastrointestinal disorders or diabetes ; and omnivorous eating habits ( including meat and dairy products more than twice per week ) , limit alcoholic intake to 1 drink ( 12  g alcohol)/week , and no oral antibiotics taken in the previous 3  months . women agreed to eat their normal diet , avoiding seaweeds and to continue their habitual use of medications , vitamins , and supplements during the study . women were recruited through the palmetto health tumor registry , the south carolina comprehensive breast center registry , and the psychosocial oncology patient database of women who had indicated their interest in participation in bc research projects and had signed consent forms giving permission to be contacted . once contacted , if eligible for study , the woman was mailed an informed consent form along with a confirmation letter of her scheduled appointment . three women dropped out of the study after the initial contact but before commencing the study , leaving 15 healthy postmenopausal women . all 15 were mammographically negative for bc ; five had never been diagnosed for bc ( controls ) , five had been treated for early ( stages 0 , i , or ii ) estrogen receptor - positive bc ( er+ ) , and five had been treated for early estrogen receptor negative bc ( er ) . diagnosis of estrogen receptor status was confirmed by the treating physician . this single - blinded placebo - controlled study incorporated a 1-month placebo run in period ( p1 ) followed by a 1-month treatment with seaweed ( s ) followed by a 1-month placebo washout period ( p2 ) . all participants ingested 5  g day of the allocated treatment in ten 500-mg capsules . at each clinic visit , women were asked about any significant health changes , including new medications or alcoholic beverages consumed . undaria pinnatifida ( harvey ) suringar ( japanese wakame ) is one of the most popular dietary seaweeds eaten in japan and korea ( maruyama et al . the u. pinnatifida was harvested by soriano from the bahia bustamante cove on the patagonian coast of argentina . we only used the sporophylls ( japanese mekabu ) , known to contain the highest amount of fucoidan ( 8  % ; soriano s.a . fucoidan is one of the water soluble components of seaweed that has shown high antitumor effects studies ( cumashi et al . a 5-g serving of undaria contained 21  kj , 0.5  g protein , 0.4  g fat , 0.06  g carbohydrate , 2.55  g fiber , 269.6  mg potassium , and 153.2  mg sodium ( soriano 2008 ) . maltodextrin ( corn products , westchester , il ) was used as placebo , and 5  g / day provided 84  kj . undaria was tested for escherichia coli , salmonella , staphylococcus aureus , pseudomonas aeruginosa , all of which were non - detectable , yeast and mold , aerobic recount , and moisture , met all safety standards ( proanalsis s.a . and food control s.a . the seaweed was milled to a 300-m powder ( done on site at gaiman by soriano s.a . , both the maltodextrin and seaweed were encapsulated into identical brown gelatin capsules ( vicofer , argentina ) . blood samples were drawn from fasting participants between 0630 and 1030 by venipuncture using sst vacutainer tubes . the blood was allowed to clot at 4  c for 30  min , and then centrifuged at 1,500 rcf for 10  min before being aliquoted into 1  ml tubes . first morning urine samples were collected on the day of each clinic visit and aliquoted into 1  ml tubes ( 200  l each ) . all samples were stored at 80  c until analyses . the iodine research laboratory , department of endocrinology , diabetes and nutrition , boston university school of medicine analyzed the capsules for iodine content . iodine in urine , empty capsules , and finished capsules was analyzed using the ceritic - arsenic redox reaction . samples were analyzed according to standard determination of total iodine protocol as outlined previously ( benotti et al . first , iodine was precipitated with perchloric acid , and the samples were digested with chloric acid . they were then measured spectrometrically at 420  nm ( autoanalyzer , technicon instrument , inc ) . serum samples were analyzed for fsh at the palmetto health richland hospital core laboratory in columbia , sc using standard direct chemiluminescent technology . urinary upar was analyzed at the university of south carolina cancer research center using upar immunoassay kit according to the manufacturer directions ( r&d systems , minneapolis , usa , cat no . the optical density was determined using a microtiter plate reader set at 450  nm ( packard spectracount , usa ) . samples were thawed and then prepared by adding 180  l of denaturing buffer ( 8  m urea , 2  % chaps , 50  mm tris , ph  9 ) to 20  l of serum then vortexed for 30  min at 4  c and diluted in 100  mm ammonium acetate ( ph  4 , 1:10 ) binding buffer . fifteen microliters of sample was added to each spot and incubated for 1  h followed by a series of washes with binding buffer and deionized h2o . samples were thawed and vortexed briefly and then centrifuged at 10,000 rcf for 5  min at 4  c . 160  l of each sample was mixed with 60  l of denaturing buffer ( 8  m urea , 2  % chaps , 50  mm tris , ph  9 ) in 1.5  ml tubes then vortex and incubated for 30  min at 4  c . the ph was then set to 4.0 using nh4oh and h3po4 . seldi cm10 ( weak cation exchange ) chip was then equilibrated with 100-l binding buffer ( 100  mm nh4oac ) and shook for 5  min . sample mix ( 200  l ) was then combined with 200-l binding buffer and vortexed briefly . binding buffer was then discarded from chips and 190-l sample mix / binding buffer was added to two spots for replication . chips were then covered with parafilm and shaken for 1  h. the samples were then removed from chips , washed three times with 100-l binding buffer and shaken for 5  min before they were washed twice with 100  l ddh2o , shook for 2  min and air dried . that was followed by applying 1  l energy absorbing material ( spa ) then dried twice . samples were analyzed at the molecular epidemiology and toxicology laboratory at the university of california , berkeley . cm10 proteinchip arrays were purchased from ciphergen biosystems ( fremont ) and prepared as described elsewhere ( hegedus et al . 2005 ) and then analyzed using seldi - tof - ms proteinchip reader ( series pbsii , ciphergen biosystems ) with laser intensity setting of 200 ( arbitrary units ) , and detector sensitivity at 8 , collecting peak data at every five positions along the spot surface . the m / z value for each of the peaks was determined using external calibration with known standards using [ arg8]-vasopressin ( 1,084.25  +  1h ) , somatostatin ( 1,637.9  +  1h ) , dynorphin a [ 209225 ] ( porcine ; 2,147.5  +  1h ) , acth [ 124 ] ( human ; 2,933.5  +  1h ) , insulin b - chain ( bovine ; 3,495.94  +  1h ) , insulin ( human recombinant ; 5,807.65  +  1h ) , and hirudin ( recombinant ; 6,963.52  +  1h ) . the ciphergen express ( ciphergen biosystems ) software collected peak data , and each peak was analyzed according to the following parameters : first pass of 5 , second pass of 2 , 0.8  % peak width named same peak , and 25  % min all spectra . our statistical models used all available data without imputation , allowing us to use the standard intention - to - treat approach ( rothman 1998 ) . to test the main study hypotheses , a repeated measures analysis of variance ( anova ) was conducted using repeated measures anova ( graphpad prism 5 ) with tukey s multiple comparison tests to determine differences between treatment groups . statistical analysis clusters were generated by ciphergen protein chip software version 3.1 ( bio - rad ) from 1.5 to 25  kda with the following settings : first - pass signal - to - noise ratio ( s / n ) of 5 , second - pass s / n of 2 , minimum peak threshold of 25  % , and a cluster mass window of 0.8  % . individuals in each group of subjects ( control , er+ , or er ) were paired for analysis by group then by treatment arm ( p1 , s , and p2 ) . mann demographic variables for the study participants are presented ( table  1 ) and show no significant differences . the health variables in table  2 show cancer survivors had higher screening fsh when compared with controls , indicating possible hormonal differences . it was also notable that the er+ had lower bmi than the other two groups.table 1demographic variables for healthy postmenopausal women at baseline compared by disease statuscontroler+ern555age years ( 95  % ci)59.8 ( 5664)60.4 ( 5566)58 ( 5066)ethnicityafrican american204european american351educationhigh school101some college or technical degree121bachelors or higher333family income ( annual)$2049,000211$5099,000213>$100,000120do not know011table 2comparison of health variables for healthy postmenopausal women at baseline compared by disease statuscontrolser+erscreening fsh ( miu ml ; 95  % ci)57.4 ( 45.4 , 69.4 ) 72.5 ( 34.4 , 110.7 ) 78.3 ( 59.5 , 97.1 ) systolic blood pressure ( mmhg ; 95  % ci)134.6 ( 119.3 , 149.9 ) 112.4 ( 97.6 , 127.2 ) 127.2 ( 114.9 , 139.5 ) diastolic blood pressure ( mmhg ; 95  % ci)96.6 ( 87.1 , 106.1 ) 74.2 ( 63.5 , 84.9 ) 89.8 ( 82.0 , 97.6 ) bmi ( 95  % ci)34 ( 27 , 41 ) 24 ( 18 , 30 ) 32 ( 23 , 41 ) bmi body mass index = weight ( kg)/height ( m)different superscript letters within each column indicate significant differences between groups ( p  =  0.05 ; i.e. , a vs. b is significant whereas a vs. a is not ) demographic variables for healthy postmenopausal women at baseline compared by disease status comparison of health variables for healthy postmenopausal women at baseline compared by disease status bmi body mass index = weight ( kg)/height ( m ) different superscript letters within each column indicate significant differences between groups ( p  =  0.05 ; i.e. , a vs. b is significant whereas a vs. a is not ) the iodine content in each 0.5  g capsule ( n  =  4 ) averaged 38.5  g . urinary iodine excretion standardized for creatinine ( in g dl g of creatinine ) increased slightly during seaweed supplementation , from 0.04 ( 95  % ci , 0.020.06 ) after p1 to 0.05 ( 95  % ci , 0.030.06 ) on s returning to 0.03 ( 95  % ci , 0.020.04 ) on p2 , indicating adherence to the protocol , but also that this low iodine - containing seaweed had little effect on iodine metabolism . these trends were the same for women taking thyroid hormone medication ( n  =  5 ) and those not taking thyroid medications ( n  =  10 ) . urinary creatinine - standardized upar concentrations decreased by almost 50  % following seaweed supplementation that was reversed after the second placebo treatment . the upar concentrations ( in pg ml g of creatinine ) were p1 , 1.5 ( 95  % ci , 0.92.1 ) ; s , 0.9 ( 95  % ci , 0.61.1 ) ; and p2 , 1.7 ( 95  % ci , 1.22.2 ; p  =  0.01 , anova ) . these trends were observed in each group , regardless of bc history ( fig . 1urinary upar concentrations compared by bc history and treatment group ( error bars indicate 95  % ci ) urinary upar concentrations compared by bc history and treatment group ( error bars indicate 95  % ci ) the seldi - ms - tof data identified a total of 14 proteins that were differentially expressed in serum ( fig .  2 ) . only one serum protein reversibly changed in all three groups with seaweed supplementation ( m / z 8,927 ) . 3 ) , and only one ( m / z 9,776 ) showed a reversible response to seaweed , increasing slightly , but significantly.fig . 2comparison of differentially expressed serum proteins ( m / z ) and their relative intensities by bc history and treatment groupfig . 3comparison of differentially expressed urine proteins ( m / z ) and their relative intensities by bc history and treatment group comparison of differentially expressed serum proteins ( m / z ) and their relative intensities by bc history and treatment group comparison of differentially expressed urine proteins ( m / z ) and their relative intensities by bc history and treatment group we first proposed that seaweed intake could be a factor in bc prevention in 1981 ( teas 1981 ) , and although the evidence from hundreds of research studies have supported seaweed as important in a host of biological pathways . our study provides evidence that 5  g day of brown seaweed ( undaria ) stimulated significant changes in urinary upar and slight changes in two other proteins . for the relatively small amount of seaweed ( 5  g day ) to result in a 50  % reduction in upar is remarkable . if supported in other studies , it gives a clear mechanism for explaining the various preventive and treatment - related effects of seaweed supplementation . at the very least , it gives a plausible mechanism for explaining the diverse findings of other studies , which include cell adhesion and detachment from the ecm of cancer cell leading to migration and metastasis ( hildenbrand and schaaf 2009 ) . in many ways , it is difficult to imagine how a mere 5  g day could have such a profound effect on systemic concentrations of upar and specific protein expression . one possibility is that seaweed , like other dietary fibers , is known to increase the mucus bilayer that protects the colonic mucosa , stimulating mucus secretion and thickness ( brownlee et al . other suggested consequences of dietary seaweed fiber include increased stool bulk which would provide protection from dietary toxins and carcinogens and reduction in amylase activity leading to decreased glycemic response . it is interesting that powdered seaweed ( 1  % , w / v ) but not seaweed extracts were responsible for almost 100  % inhibition of amylase and pepsin digestive enzyme activity in vitro ( bobin - dubigeon et al . small water - soluble molecules which would be lost in the production of seaweed extracts were thought to be responsible for the inhibition of protease activities . thus , the small amount of dietary seaweed used in our study and consumed in japan might be expected to have a profound effect in humans . our study also provides a possible rationale for understanding why bc in non - seaweed - consuming western populations increases with age , but remains more or less stable for japanese women ( reddy et al . upar increases after menopause ( chung et al . 1998 ) , and if seaweed , on a population level in japan , has a significant impact on upar concentrations , it may be the basis for the stable rates . several of proteins with high intensity have also been identified as important in bc ( gast et al . upar has an m / z protein size beyond the range we measured , but two proteins we identified changed with seaweed . serum protein ( m / z 8,927 ) has been identified as complement activation c3a des - arginine and may indicate stimulation of an immune response that has also been detected in subjects with early bc ( becker et al . enhanced immune function has been reported in vitro ( kim and joo 2008 ) and in vivo ( kandasamy et al . 2012 ; leonard et al . 2012 ) . increased expression of m / z 8,928 may be associated with the observed enhanced immune function . the urinary protein m / z 9,776 has not been identified as important in bc but it has been identified as a hormone - responsive protein important in decidualization of the endometrial stromal matrix preceding embryo implantation ( oh et al . this progesterone - responsive protein increased with seaweed . in a study of conception rates in heat - stressed cows , seaweed supplementation with a different brown seaweed ( ascophyllum ) was associated with more than a 7-fold increase in successful pregnancies ( kellogg et al . this suggests that seaweed supplementation may influence cell surface signaling receptors for steroid hormones . as bc in japan is characterized by higher rates of the more favorable er+ type , we anticipated that women with a history of er bc might have different protein responses to dietary seaweed . there were only minor differences in protein expression by bc history , and possibly the variations might reflect underlying differences in bc susceptibility . further research should be conducted to define whether or not these minor differences reflect meaningful biological variations . seaweeds are consumed daily in japan and are considered to be important in maintaining optimal health . several different kinds of seaweed are eaten daily in a variety of forms and dishes and are consumed dried in sushi wrappers , fresh or rehydrated as a side dish , and used as flavor condiments as well as the basis for broth - based ( dashi ) flavoring added to other foods . our best guess is that 5  g / day may approximate the mean japanese daily intake . a typical serving of the kind of seaweed used in this study , wakame ( undaria ) , has been estimated at 1  g by the japanese ministry of health ( ministry of health of japan 2004 ) . thus our study could overestimate the contribution of undaria to the average japanese consumer , but as a pilot study , we propose that it combines the mean dose of seaweed and isolates a particular kind of seaweed , so the effects we report can be attributed to one seaweed species . our primary reason for using whole seaweed rather than a purified fraction is that people in japan consume seaweed , and it is this dietary preference that appears to be associated with multiple health benefits ( macartain et al . the combination of various bioactive components in whole seaweed may be greater than the sum of the individual parts , were they to be purified and tested individually . many previous in vitro and in vivo studies have used hot or cold water extracts of undaria , and have proposed that fucoidan , a water - soluble sulfated polysaccharide primarily found in brown seaweeds , as the most bioactive component ( cumashi et al . in previous studies we have demonstrated that undaria fucoidan is absorbed by humans , despite its large molecular size ( irhimeh et al . although it would be interesting to have used such an extract in our study , it would have required obtaining approval to use the extract as an investigational new drug , which would have taken extensive and costly pretesting evaluation to register with the us food and drug administration . as an alternative , we chose to use high - fucoidan seaweed , using only the sporophyll of undaria , collected at the peak of the summer harvest season . these sporophylls consistently contain 8  % fucoidan / dry weight and are also low in iodine ( approximately 80  g g ) , and 5  g day would be well under the tolerable upper intake level of 1,100  g / day ( national research council 2001 ) . in addition , no adverse effects have been recorded in our previous studies using this 5  g day dose of undaria sporophylls ( teas et al . one of our study strengths was that it evaluated the introduction of seaweed to a non - seaweed - consuming population , and despite the women continuing with their normal diet , significant changes in protein expression were observed . the reversibility of the changes in the urinary concentration of upar and of the two seldi - identified proteins strongly suggests the changes were attributable to the inclusion of seaweed . as any small proof of principle study , our small sample size was a limitation . . data from upar concentration changes indicate that seaweed affects ecm , which is known to influence cell surface signaling , cell adhesion , and growth factor communication and responsiveness in breast tissue . additional data from seldi proteomic analysis further supports the role of seaweed supplementation in modulating proteins known to affect cell adhesion , immune response , and inflammatory responses . these effects may be mediated through the ecm and be important in maintaining healthy structure and function in breast cells . in particular , lower upar concentrations could reduce the likelihood of breast tissue transformation and cancer cell proliferation and invasiveness . the role of dietary seaweed in lowering upar may therefore be critical in explaining differences in postmenopausal bc incidence and mortality between japanese women and women in other developed countries . the possibility that dietary seaweed could decrease upar is especially important in that there are no anti - upar drugs currently available ( degryse 2011 ) . finding that seaweed can have a significant effect on upar could be helpful both in designing future drugs and dietary interventions for people with cancer .
daily consumption of seaweed has been proposed as a factor in explaining lower postmenopausal breast cancer ( bc ) incidence and mortality rates in japan . 
 this clinical trial assessed the impact of introducing seaweed- to non - seaweed - consuming american postmenopausal women . 
 fifteen healthy postmenopausal women were recruited for a 3-month single - blinded placebo controlled clinical trial ; five had no history of bc ( controls ) and ten were bc survivors . 
 participants ingested ten capsules daily ( 5  g day1 ) of placebo for 4  weeks , seaweed ( undaria ) for 4  weeks , then placebo for another 4  weeks . 
 blood and urine samples were collected after each treatment period . 
 urinary human urokinase - type plasminogen activator receptor concentrations ( upar ) were analyzed by elisa , and urine and serum were analyzed for protein expression using surface - enhanced laser desorption / ionization  time - of - flight mass spectrometry ( seldi - tof - ms ) . 
 urinary creatinine standardized upar ( in pg ml g1 creatinine ) changed significantly between groups , decreasing by about half following seaweed supplementation ( placebo 1 , 1.5 ( 95  % ci , 0.92.1 ) and seaweed , 0.9 ( 95  % ci , 0.61.1 ) while placebo 2 returned to pre - seaweed concentration ( 1.7 ( 95  % ci , 1.2 - 2.2 ) ; p  =  0.01 , anova ) . 
 one seldi - tof - ms - identified urinary protein ( m / z 9,776 ) showed a similar reversible decrease with seaweed and is reported to be associated with cell attachment . 
 one serum protein ( m / z 8,928 ) reversibly increased with seaweed and may be the immunostimulatory complement activation c3a des - arginine . 
 upar is higher among postmenopausal women generally , and for bc patients , it is associated with unfavorable bc prognosis . by lowering upar , dietary seaweed may help explain lower bc incidence and mortality among postmenopausal women in japan .