Isoflavones in human breast milk and other biological fluids.

We established a method for using HPLC and diode-array ultraviolet scanning to quantitate soy isoflavonoids in foods and in human plasma, urine, and breast milk. The analytes occurring as glycoside conjugates were hydrolyzed enzymatically before HPLC analysis if extracted from biological matrices or were subjected to direct HPLC analysis after extraction from foods. We monitored the isoflavones daidzein, genistein, glycitein, formononetin, and biochanin-A and their mammalian metabolites equol and O-desmethylangolensin in human plasma, urine, and breast milk. Analytes were identified by absorbance patterns, fluorometric and electrochemical detection. and comparison with internal and external standards. In addition, we identified analytes by using gas chromatography-mass spectrometry after trimethylsilylation. The HPLC method was also used to measure concentrations of isoflavones and their glucoside conjugates in various soy-based infant formulas. Total isoflavone concentrations varied between 155 and 281 mg/kg. After one woman received a moderate challenge with 20 g roasted soybeans (equivalent to 37 mg isoflavones), we detected mean total isoflavone concentrations of approximately 2.0 micromol/L in plasma, 0.2 micromol/L in breast milk, and 3.0 micromol/h in urine. According to our measurements, with adjustment for body weight, isoflavonoid exposure is 4-6 times higher in infants fed soy-based formula than in adults eating a diet rich in soyfoods (approximately 30 g/d). Implications of the presented results for the potential cancer-preventing activity of isoflavones by exposing newborn infants to these phytochemicals are discussed.

Seasonal variations in plasma micronutrients and antioxidants.

Plasma samples were collected at monthly intervals for a period of 1 year from a group of healthy nonsmoking men and women (n = 21) living in Honolulu, HI. Analysis of plasma cholesterol and triglyceride levels showed marked seasonal variations, with higher mean levels in winter months and lower values in the summer. Cholesterol and triglycerides were highly and inversely correlated with plasma levels of the provitamin A carotenoids. Mean beta- and alpha-carotene levels were highest in late summer and fall. Plasma retinol levels were significantly lower in the summer and higher in the winter. Variations (either between individuals or seasonally) in plasma retinol were unrelated to plasma provitamin A carotenoid levels. Plasma levels of alpha-tocopherol, gamma-tocopherol, beta-cryptoxanthin, and lutein were also higher in the winter and lower in the summer. Significant seasonal correlations, both positive and negative, with environmental variables, such as temperature, solar UV radiation, and rainfall, are noted for many of these plasma micronutrients. The number of samples required to accurately characterize long-term plasma levels for an individual generally ranged from 1 to 4. However, plasma retinol levels exhibited the highest ratio of intra- to interindividual variability, suggesting the need for multiple sampling (> 8 samples) for this micronutrient. Some of this variability for retinol was associated with seasonal changes. Assessment by a diet history of food and supplement intake of micronutrients and phytochemicals for 1 year showed good agreement with 1-year mean plasma levels for most carotenoids, vitamin C, and alpha-tocopherol. Retinol, gamma-tocopherol, cholesterol, and triglyceride levels in plasma were unrelated to estimates of dietary intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlations between mutagen sensitivity and plasma nutrient levels of healthy individuals.

Increased mutagen sensitivity and decreased intake of antioxidant-rich fruits and vegetables have been associated with an increased risk of upper aerodigestive tract cancers. The objective of this study was to investigate the intraindividual variation in mutagen sensitivity and its possible correlation with plasma nutrient levels in a group of 25 healthy individuals in Hawaii. Mutagen sensitivity, as assessed by bleomycin-induced chromosomal breaks in cultured peripheral blood lymphocytes and plasma nutrient levels were measured monthly for 11 months. The monthly numbers of chromosomal breaks/cell ranged from 0.04 to 0.80 and showed considerable intraindividual variation. Based on individual means, significant inverse correlations were found between mutagen sensitivity scores and the plasma levels of alpha-carotene (r = -0.64), total carotenoids (r = -0.41), and ascorbic acid (r = -0.40). There were also significant inverse associations between monthly mean plasma levels of alpha-carotene (r = -0.58), beta-carotene (r = -0.76) and total carotenoids (r = -0.72) and monthly mean chromosomal breaks. In contrast, there was a significant positive correlation between monthly mean plasma triglyceride level (r = 0.60) and monthly mean mutagen sensitivity. These results suggest that mutagen sensitivity as assessed by the bleomycin assay may be influenced by plasma levels of certain nutrients and could potentially be modified by dietary interventions or micronutrient supplementation.

Predictors of N-acetyltransferase activity: should caffeine phenotyping and NAT2 genotyping be used interchangeably in epidemiological studies?

To determine whether NAT2 genotyping could be used interchangeably with caffeine phenotyping in assessing N-acetyltransferase activity in epidemiological studies, sources of interindividual variability in N-acetyltransferase activity were assessed among 90 subjects of various ethnic backgrounds in Hawaii. Forty-three subjects were patients with in situ colorectal cancer treated by polypectomy, and 47 were healthy population controls. Subjects were administered a lifestyle questionnaire and were evaluated for N-acetyltransferase activity by caffeine phenotyping. NAT2 genotype was also assessed by PCR amplification of peripheral leukocyte DNA for the M1, M2, and M3 variant alleles. Fifty-four % of the overall variation in acetylation activity was explained by the three genotype categories (homozygous variant, heterozygous, and homozygous wild-type). This proportion was reduced to 42% when genotype was modeled using only two categories ("slow" being homozygous variant; "rapid" being all others). Use of gout medications (probenecid or allopurinol), consumption of heavily browned fish, and P450IA2 activity (also measured by caffeine phenotyping), together explained another 11% of the variance. No association was found between acetylation activity and sex; race; age; education; smoking; physical activity; weight; consumption of coffee, alcohol, red meat, processed meat, and cruciferous vegetables; or use of menopausal estrogens, after taking genotype into account. Results were similar for colorectal cancer patients and controls. Considerable variation in acetylation activity was observed within the homozygous wild-type group. This study suggests that the use of genotyping, instead of phenotyping, to assess the association of acetylation with cancer risk is unlikely to introduce major misclassification or bias, especially when the three genotype categories are modeled and the sample size is large. However, when the rapid acetylation phenotype is the at-risk group (e.g., when studying colon career), phenotyping appears judicious given the variability in acetylation activity within this group.

Urinary excretion of isoflavonoids and the risk of breast cancer.

Isoflavonoids are a group of biologically active phytochemicals that humans are exposed to mainly through soy food intake. Because of the similar chemical structure of these compounds and estradiol, it has been hypothesized that isoflavonoids may be related to the risk of breast cancer. Overnight urine samples from 60 incident breast cancer cases and their individually matched controls were assayed for urinary excretion rates of five major isoflavonoids (daidzein, genistein, glycitein, equol, and O-desmethylangolensin) and total phenols. These subjects were from a large population-based case-control study conducted in Shanghai, and urine samples from breast cancer cases were collected before any cancer therapy to minimize the potential influence of the disease and its sequelae on study results. Urinary excretion of total phenols and all individual isoflavonoids, particularly glycitein, was substantially lower in breast cancer cases than controls. For total isoflavonoids, the mean excretion was 13.95 nmol/mg creatinine (SD, 20.76 nmol/mg creatinine) for cases and 19.52 nmol/mg creatinine (SD, 25.36 nmol/mg creatinine) for controls (P for difference = 0.04). The case-control difference was more evident when median levels of these compounds were compared, with the median excretion of all major isoflavonoids being 50-65% lower in cases than in controls. Individuals in the highest tertile of daidzein, glycitein, and total isoflavonoids had about half the cancer risk of those in the lowest tertile. The adjusted odds ratio for breast cancer was 0.14 (95% confidence interval, 0.02-0.88) for women whose urinary excretion of both phenol and total isoflavonoids was in the upper 50% compared with those in the lower 50%. The results from this study support the hypothesis that a high intake of soy foods may reduce the risk of breast cancer.

Combined effects of well-done red meat, smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk.

Heterocyclic amines (HAAs) are suspected carcinogens that are formed in meat when it is cooked at high temperature for long durations. These compounds require metabolic activation by CYP1A2 and N-acetyltransferase (NAT) 2 or NAT1 before they can bind to DNA. It has been hypothesized that well-done meat increases the risk of colorectal cancer (CRC), especially in individuals with the rapid phenotype for CYP1A2 and NAT2. This association may be particularly strong in smokers because smoking is known to induce CYP1A2. We conducted a population-based case-control study on Oahu, Hawaii to specifically test this hypothesis. An in-person interview assessed the diet and preference for well-done red meat of 349 patients with CRC and 467 population controls. A urine collection after caffeine challenge and a blood collection were used to assess phenotype for CYP1A2 and NAT2 and genotype for NAT2 and NAT1, respectively. No statistically significant main effect association with CRC was found for red meat intake, preference for well-done red meat, the NAT2 rapid genotype, the CYP1A2 rapid phenotype or the NAT1*10 allele. However, in ever-smokers, preference for well-done red meat was associated with an 8.8-fold increased risk of CRC (95% confidence interval, 1.7-44.9) among subjects with the NAT2 and CYP1A2 rapid phenotypes, compared with smokers with low NAT2 and CYP1A2 activities who preferred their red meat rare or medium. No similar association was found in never-smokers, and there was no increased risk for well-done meat among smokers with a rapid phenotype for only one of these enzymes or for smokers with both rapid phenotypes who did not prefer their red meat well-done. These data provide additional support to the hypothesis that exposure to carcinogens (presumably HAAs) through consumption of well-done meat increases the risk of CRC, particularly in individuals who are genetically susceptible (as determined by a rapid phenotype for both NAT2 and CYP1A2) and suggest that smoking, by inducing CYP1A2, facilitates this effect.

Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids.

Plants are more susceptible to the toxic effects of nitrogen dioxide when exposure takes place in the dark. Beta-carotene and other common carotenoids react with nitrogen dioxide in the dark to yield intermediate nitrosating agents consistent with the formation of nitrate esters. Simultaneous exposure of carotenoids to NO2 and light significantly reduced formation of nitrosating intermediates and resulted in the release of nitric oxide (NO) into the gas phase. Light-mediated reduction of NO2 to NO by carotenoids may be an important mechanism for preventing damage in plants exposed to NO2. The formation of nitrosating agents from the reaction of carotenoids with NO2 suggests that their ability to prevent nirosative damage associated with NO2 exposure in both plants and animals may be limited in the absence of light.

Isoflavone levels in soy foods consumed by multiethnic populations in Singapore and Hawaii.

Concentrations and glucosidic conjugation patterns of isoflavones were determined in soy foods consumed by multiethnic populations in Singapore and Hawaii. Six raw and 11 cooked food groups traditionally consumed in Singapore and 8 food groups consumed in Hawaii were analyzed by reversed-phase high-pressure liquid chromatography with diode array detection. Mean total isoflavone levels varied between 35 and 7500 ppm, with the lowest values found in soy milk and burgers and the highest levels observed in soybean and its seeds and in supplements. Total isoflavone levels and conjugation patterns varied as a function of soybean variety, storage conditions, and food processing. A large contribution to the differences in total isoflavone content between food groups was due to the water content in foods and to leaching of polar analytes into the water phase during boiling. Soy protein drinks and traditional soy foods were found to possess very similar isoflavone amounts considering usual serving sizes.

Inhibition of neoplastic transformation and bioavailability of dietary flavonoid agents.

Evaluation of unknown biological effects of chemicals including food plant products requires the assessment of bioactivity and bioavailability. Epidemiologic studies show consistently a cancer protective effect of fruit and vegetable consumption, but there is little understanding of which phytochemicals account for this observation. Commonly studied antioxidant micronutrients are less consistently correlated with cancer protection relative to the food groups themselves, suggesting that other phytochemicals or a combination of food products play key roles in preventing cancer. We investigated the effects of the predominant dietary flavonoids and isoflavonoids at inhibiting neoplastic transformation induced by 3-methylcholanthrene in C3H 10T1/2 murine fibroblasts. We found that most phenolic agents tested were equal to or superior to known chemopreventive agents such as carotenoids or vitamins in effectiveness. Hesperetin, hesperidin and catechin were the most potent agents among the flavonoids tested, inhibiting transformation completely when applied at 1.0 microM after exposure to the carcinogen. Structure-activity comparison revealed that among the compounds tested, flavonoids with a vicinal diphenol structure in ring 'B' and a saturated 'C' ring exhibited the strongest effects. Most agents tested showed dose-dependent patterns. Interestingly, the soy isoflavonoids were weakly active except when applied in combination, suggesting a synergistic effect. In addition, HPLC techniques were developed for determining the bioavailability of isoflavonoids in human biological fluids including urine, plasma and breast milk. We observed a relatively fast absorption, distribution and elimination of isoflavonoids including a biphasic pattern probably due to enterohepatic circulation. Total peak isoflavone levels in urine, plasma and in breast milk were found to be 60 microM, 2 microM and 0.2 microM, respectively and were reached 8-12 hours after consumption of soy foods. Levels detected in human body fluids were found to be highly active at inhibiting neoplastic transformation, especially considering synergistic effects observed for combinations of daidzein and genistein, the predominant isoflavonoids occurring in soy foods.

Application of HPLC coupled with ultraviolet-photodiode array detection for the analysis of phytoestrogens in biological samples.

HPLC/PDA methodology was developed for phytoestrogen analysis in human blood, urine, and breast milk. Applications of this technique in four different observational studies showed that urinary excretion rates of isoflavonoids reflects soy consumption reliably. A case-control study in Shanghai showed a decrease in risk to develop breast cancer for women with high urinary isoflavone excretion. These findings support a potential breast cancer preventive effect achieved by soy consumption in populations that eat soy foods habitually.

Coenzyme Q10, carotenoid, tocopherol, and retinol levels in cord plasma from multiethnic subjects in Hawaii.

Coenzyme Q10 (Q10), carotenoids, tocopherols, and retinol are the major circulating lipid-phase micronutrients (LPM) known to help mitigate oxidative damage and prevent chronic diseases. However, the functions of these compounds in newborns are little understood. This is due, in part, to the paucity of studies reporting their concentrations in this population. We measured Q10, carotenoids, tocopherols, and retinol in cord plasma from 100 multiethnic subjects living in Hawaii using HPLC with diode array and electrochemical detection. Appropriate internal standards were used including, for the first time, custom designed oxidized (UN10) and reduced (UL10) Q10 analogues. These compounds reflected the oxidation of UL10 to UN10 that occurred during sample processing and analysis and thus permitted accurate adjustments of natively circulating Q10 levels. All LPM measured were much lower in cord than in peripheral plasma. Cord plasma levels of total carotenoids, tocopherols, and retinol were approximately 10-fold, 3- to 5-fold and 1.5- to 3-fold lower than those in children or women. Cord plasma levels of total Q10 (TQ10; median, 113 ng/mL) were approximately 2-fold or 7- to 9-fold lower than peripheral plasma levels of neonates or children and adults, respectively. In contrast, the UN10/TQ10 ratio was substantially higher in cord (24%) than in peripheral plasma of children (3-4%) or adults (9%). Among the 5 ethnic groups in our cohort, no differences were observed in the levels of UN10, UL10, or TQ10. However, significant differences in many of the LPM were observed between ethnicities. More research is needed to explain these phenomena.

High-performance liquid chromatographic assay of isoflavonoids and coumestrol from human urine.

A rapid, sensitive and precise diode-array reversed-phase HPLC method was developed for human urine analysis of the most common dietary isoflavones daidzein, genistein, formononetin and biochanin-A, their mammalian metabolites equol and O-desmethylangolensin, and of coumestrol, another commonly occurring phytoestrogen. Analytes were isolated and concentrated by solid-phase extraction and separated by HPLC followed by identification through retention times and UV scans, and in the case of coumestrol additionally by fluorometric response. This method was applied to monitor changes in urinary excretion of these analytes after challenge with soybeans and was evaluated for precision and recovery of analytes.

Daidzein and genistein concentrations in human milk after soy consumption.

Soy isoflavones were quantified from human milk by a fast, precise, and selective HPLC method after enzymatic hydrolysis of conjugated isoflavones and extraction with ethyl acetate. Isoflavone aglycones and their mammalian metabolites equol and O-desmethylangolensin were separated selectively and identified by absorbance patterns, fluorometric and electrochemical detection, gas chromatography-mass spectrometric analysis after trimethylsilylation, and with internal and external authentic standards. HPLC injections of 20 microL of human milk showed detection limits of 1-3 pmol for all analytes by using diode-array detection. The detection limit could be improved by as much as 1000-fold by extended concentration through partitioning with ethyl acetate, by using electrochemical detection, by increasing the injection volumes, or by combining these techniques. We used the proposed method to monitor isoflavone concentrations in human milk and in human urine after challenge with 5, 10, and 20 g of roasted soybeans in the diet. Implications of the results for the potential of isoflavones to prevent cancer in newborn infants exposed to these agents are discussed.

Synthetic carotenoids as internal standards for plasma micronutrient analyses by high-performance liquid chromatography.

A method is described for the synthesis of new carotenoids by transesterification of ethyl-beta-apo-8'-carotenoate. The rate of transesterification was temperature-dependent with highest yields using primary alcohols. Reaction conditions were found avoiding Z isomerization. The structures of the new products were confirmed by ultraviolet, mass, 1H and 13C nuclear magnetic resonance spectroscopy. The synthetic carotenoids were used as internal standards for reversed-phase high-performance liquid chromatographic (HPLC) analyses of lipid phase plasma micronutrients. Retention times for the trans-esterified products increased as a function of alcohol chain length allowing a choice of synthetic carotenoids for use as internal standards for carotenoid quantitation without interference with the detection of analytes during HPLC analysis.

Rapid HPLC analysis of dietary phytoestrogens from legumes and from human urine.

Due to growing evidence suggesting that phytoestrogens might protect against various cancers, particularly against breast and prostate cancer, it is important to measure the exposure of populations to these compounds by determining levels in food and in human tissue or body fluids to assess the possible cancer protective properties of these agents. Therefore, we developed a simple and fast procedure to extract and simultaneously hydrolyze phytoestrogens and their conjugates from food items, and present a fast and selective high-performance liquid chromatography (HPLC) method for precise determinations of the most common dietary phytoestrogens genistein, biochanin-A, daidzein, formononetin, and coumestrol using flavone as internal standard. For the first time HPLC was applied to measure these phytoestrogens and their most abundant metabolites equol and O-desmethyl-angolensin from human urine. The proposed methodology has been evaluated for losses due to thermal degradation during extraction and hydrolysis and due to sample handling during the entire work-up including solid phase extraction, and values are given for inter- and intra-assay variability. We present isoflavonoid levels of most common peas and beans used in "western" and "eastern" diets and compare isoflavonoid and coumestrol levels of raw, canned, and cooked foods which can be used in future epidemiological studies. We also determined human urinary levels with our methodology comparing values before and after soybean intake.

HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids.

A fast, precise and selective diode array HPLC method is presented for the extraction and analysis of soy isoflavonoids from foods and from human urine, plasma, and breast milk in support of mechanistic and epidemiologic studies assessing the potential cancer protective role of soya or isoflavones. Solid phase or solvent extraction was chosen for isolation, and enzymatic or acid hydrolysis procedures were used for aglycone production depending on the matrix to be analyzed. C-18 reversed-phase HPLC was applied to selectively separate and quantitate daidzein (1), glycitein (3), and genistein (4), including their malonyl (a) and acetyl (b) esters, and their mammalian metabolites equol (6) and O-desmethylangolensin (7), as well as formononetin (2), biochanin-A (5), and coumestrol (8) using a gradient elution system. UV absorbance scans and authentic standards were applied for identification purposes, additional to fluorometric monitoring, electrochemical detection, and GC/ MS analysis after trimethyl silylation. Detection limits of 20-microl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and O-desmethylangolensin (DMA), respectively, by monitoring at the individual compound's absorption maximum. The proposed method was applied to monitor isoflavone levels in soy foods and in human plasma, urine and breast milk after challenge with roasted soybeans. Implications of the presented results on the potential activity of isoflavones to prevent cancer by exposing newborn infants to these agents are discussed.

Serum levels and metabolic clearance of the isoflavones genistein and daidzein in hemodialysis patients.

Genistein and daidzein are biologically active isoflavones that are especially abundant in soybeans. After intestinal absorption, circulating genistein and daidzein are eliminated primarily by the kidneys. This study was undertaken to assess the metabolism of genistein and daidzein in patients with end-stage renal disease (ESRD) on hemodialysis therapy, and to test whether this treatment modality can replace the lack of kidney function, with respect to the elimination of the isoflavones. Twenty-three hemodialysis patients and 10 healthy subjects were studied. While consuming a self-selected low isoflavone diet, baseline blood levels were undetectable in eight of 10 healthy subjects and in 14 of 23 dialysis patients. The remaining participants had detectable levels, with the nine dialysis patients displaying much higher blood concentrations than the two healthy control subjects. After the evening intake of one dose of an isoflavone-rich soy protein isolate drink, the early morning blood levels of genistein and daidzein were higher in seven dialysis patients than in eight healthy subjects (genistein 1271+/-321 versus 425+/-104, P<0.05; daidzein 1304+/-352 versus 292+/-78, P<0.05). The blood clearance of the isoflavones was studied in two healthy subjects and in three dialysis patients. Genistein and daidzein were eliminated within 2 d in the healthy subjects, but had not returned to baseline in two of three ESRD patients, 7 d after intake. The half-life of both compounds was estimated to be 10-fold longer in the ESRD patients than in the healthy subjects. Finally, genistein and daidzein levels were measured before and after dialysis in five patients, both while on their regular diet and after one dose of a soy protein isolate drink. In both instances, the dialysis treatment did not affect the blood isoflavone levels. In conclusion, approximately one-third of hemodialysis patients eating the standard American renal diet experience high blood levels of the isoflavones genistein and daidzein, while the remaining two-thirds have undetectable levels. After ingestion of isoflavone-rich food such as soy products, all patients have detectable levels that remain very high for several days due to lack of renal excretion.

Effects of dietary sesame seeds on plasma tocopherol levels.

The tocopherols, the major vitamers of vitamin E, are believed to play a role in the prevention of human aging-related diseases such as cancer and heart disease, yet little is known concerning determinants of their plasma concentrations. Evidence from animal studies suggests that the dietary source of gamma-tocopherol can significantly affect plasma levels of this tocopherol as well as its functional vitamin E activity. To determine whether plasma levels of tocopherols in humans are similarly altered, a study was undertaken in which subjects (n = 9) were fed muffins containing equivalent amounts of gamma-tocopherol from sesame seeds, walnuts, or soy oil. We observed that consumption of as little as 5 mg of gamma-tocopherol per day over a three-day period from sesame seeds, but not from walnuts or soy oil, significantly elevated serum gamma-tocopherol levels (19.1% increase, p = 0.03) and depressed plasma beta-tocopherol (34% decrease, p = 0.01). No significant changes in baseline or postintervention plasma levels of cholesterol, triglycerides, or carotenoids were seen for any of the intervention groups. All subjects consuming sesame seed-containing muffins had detectable levels of the sesame lignan sesamolin in their plasma. Consumption of moderate amounts of sesame seeds appears to significantly increase plasma gamma-tocopherol and alter plasma tocopherol ratios in humans and is consistent with the effects of dietary sesame seeds observed in rats leading to elevated plasma gamma-tocopherol and enhanced vitamin E bioactivity.

Gamma-tocopherol detoxification of nitrogen dioxide: superiority to alpha-tocopherol.

In the vitamin E group, alpha-tocopherol is generally considered to be the most potent antioxidant with the highest vitamin bioactivity, yet gamma-tocopherol is produced in greater amounts by many plants and is the principal tocopherol in the United States diet. This report describes a fundamental difference in the chemical reactivities of alpha-tocopherol and gamma-tocopherol with nitrogen dioxide (NO2), which leads to the formation of a nitrosating agent from alpha-tocopherol, but not from gamma-tocopherol. Nitric oxide (NO) is a major product of the reaction of gamma-tocopherol with NO2, while alpha-tocopherol reacts with NO2 to form an intermediate tocopheroxide analogue. The biological significance of gamma-tocopherol is suggested by limited epidemiological data as well as the observation that it is a more potent inhibitor than alpha-tocopherol of neoplastic transformation during the postinitiation phase in 3-methylcholanthrene-treated C3H/10T1/2 murine fibroblasts. This latter property suggests the superiority of gamma-tocopherol in a mammalian biological assay and a role for endogenous NO production in promotion of neoplastic transformation.

Reproducibility of erythrocyte polyamine measurements and correlation with plasma micronutrients in an antioxidant vitamin intervention study.

Erythrocyte polyamine measurements have been previously investigated as candidate biomarkers for hyperproliferation and recently as a potential intermediate endpoint in clinical chemoprevention trials with difluoromethylornithine, an inhibitor of polyamine biosynthesis. This study was performed to determine the reproducibility of erythrocyte polyamine measurements and their possible correlation with plasma micronutrients in seven healthy adults in an antioxidant vitamin intervention study. As part of this cross-over intervention study, three subjects took beta-carotene (31.4 mg/day) plus D-alpha-tocopherol acetate (720 IU/day) supplements during the first 3 months and four subjects took the supplements during the second 3 months. Heparinized blood samples were collected at baseline and every month over total 6 months for simultaneous determination of erythrocyte polyamines and plasma micronutrients by the high-performance liquid chromatographic method. For all the measures of erythrocyte polyamines the intraindividual variation was smaller than that between subjects, and three or four measurements required to accurately characterize long-term erythrocyte polyamines for an individual. The intra-class correlations were moderately high for all erythrocyte polyamine measurements, indicating a good reproducibility for intra-individual erythrocyte polyamine measurements. Based on monthly values, significant inverse correlations were found between erythrocyte spermidine and the plasma levels of retinol (r = -0.50) and lutein (r = -0.52). There were also significant inverse associations between erythrocyte spermine and plasma levels of alpha-tocopherol (r = -0.29), lutein (r = -0.44), lycopene (r = -0.29), beta-cryptoxanthin (r = -0.30), and total carotenoids (r = -0.29). The effects of supplementation upon the associations between erythrocyte polyamines and plasma nutrient levels were additionally addressed. The results indicate an acceptable longitudinal reproducibility of erythrocyte polyamine measurements, support the hypothesis that erythrocyte polyamine measurements may be correlated with plasma levels of certain nutrients, and suggest a further biomarker application in cancer prevention trials involving dietary modifications or specific relevant micronutrients.