First-morning Void Research Articles

Consumption of vegetables alters morning urinary 6‐sulfatoxymelatonin concentration

Melatonin, which is contained in certain vegetables, may have an influence on circulatory melatonin concentrations. This study examined the effects of the consumption of vegetables on 6-sulfatoxymelatonin concentrations in morning urine. Ninety-four healthy women aged 24-55 were recruited through a city public health center in Japan. The women randomly allocated to the intervention group were requested to consume high amounts of six selected vegetables, with a target of 350 g/day for 65 days, while those in the control group were asked to avoid the same six vegetables during the same period. First-void morning urine was collected before and at the end of the intervention period, and creatinine-adjusted 6-sulfatoxymelatonin concentrations were measured. At the end of the intervention period, daily mean intake of melatonin from the six vegetables was 1288.0 ng in the intervention group and 5.3 ng in the control group. In the intervention group, the mean concentration of 6-sulfatoxymelatonin changed from 48.1 [95% confidence interval (CI): 40.4-57.2] ng/mg creatinine to 49.6 (95% CI: 42.8-57.3) ng/mg creatinine across the intervention period. In the control group, the mean concentration of 6-sulfatoxymelatonin changed from 55.5 (95% CI: 48.7-63.2) ng/mg creatinine to 50.8 (95% CI: 44.0-58.7) ng/mg creatinine across the intervention period. A comparison of the two groups with regard to the changes in the 6-sulfatoxymelatonin concentrations across the intervention period showed a significant difference (P = 0.03). The results indicate that increased consumption of vegetables raises circulatory melatonin concentrations.

An observational study of the potential for human exposures to pet-borne diazinon residues following lawn applications

This study examined the potential for pet dogs to be an important pathway for transporting diazinon residues into homes and onto its occupants following residential lawn applications. The primary objectives were to investigate the potential exposures of occupants and their pet dogs to diazinon after an application to turf at their residences and to determine if personal contacts between occupants and their pet dogs resulted in measurable exposures. It was conducted from April to August 2001 before the Agency phased out all residential uses of diazinon in December 2004. Six families and their pet dogs were recruited into the study. Monitoring was conducted at pre-, 1, 2, 4, and 8 days post-application of a commercial, granular formulation of diazinon to the lawn by the homeowner. Environmental samples collected included soil, indoor air, carpet dust, and transferable residues from lawns and floors. Samples collected from the pet dogs consisted of paw wipes, fur clippings, and transferable residues from the fur by a technician or child wearing a cotton glove(s). First morning void (FMV) urine samples were collected from each child and his/her parent on each sampling day. Diazinon was analyzed in all samples, except urine, by GC-MS. The metabolite 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMPy) was analyzed in the urine samples by HPLC-MS/MS. Mean airborne residues of diazinon on day 1 post-application were at least six times higher in both the living rooms (235±267 ng/m 3) and children's bedrooms (179±246 ng/m 3) than at pre-application. Mean loadings of diazinon in carpet dust samples were at least 20 times greater on days 2, 4, and 8 post-application than mean loadings (0.03±0.04 ng/cm 2) at pre-application. The pet dogs had over 900 times higher mean loadings of diazinon residues on their paws on day 1 post-application (88.1±100.1 ng/cm 2) compared to mean loadings (<0.09 ng/cm 2) at pre-application. The mean diazinon loadings on the fur clippings were at least 14 times higher on days 1, 2, 4, and 8 post-application than mean loadings (0.8±0.4 ng/cm 2) at pre-application. For transferable residues from dog fur, the mean loadings of diazinon on the technician's cotton glove samples were the lowest before application (0.04±0.08 ng/cm 2) and the highest on day 1 post-application (10.4±23.9 ng/cm 2) of diazinon to turf. Urinary IMPy concentrations for the participants ranged from <0.3 to 5.5 ng/mL before application and <0.3–12.5 ng/mL after application of diazinon. The mean urinary IMPy concentrations for children or adults were not statistically different ( p>0.05) at pre-application compared to post-application of diazinon to turf. The results showed that the participants and their pet dogs were likely exposed to low levels of diazinon residues from several sources (i.e., air, dust, and soil), through several pathways and routes, after lawn applications at these residences. Lastly, the pet dog appears to be an important pathway for the transfer and translocation of diazinon residues inside the homes and likely exposed occupants through personal contacts (i.e., petting).

Urinary 6-Sulfatoxymelatonin and Mammographic Density in Japanese Women

A protective role of melatonin in the etiology of breast cancer has been suggested. The down-regulation of estrogen secretion by melatonin is thought to be a main cause of the link between melatonin and breast cancer risk. The present cross-sectional study examined whether the urinary 6-sulfatoxymelatonin (aMT6-s) level is inversely associated with mammographic density, which is regarded as a marker of breast cancer risk. The study subjects were 289 Japanese women (175 premenopausal and 123 postmenopausal women) who were recruited from participants in a mammographic breast cancer screening. The size of the total breast area and that of the dense area were measured quantitatively using an automated mammographic mass detection method. The concentration of aMT6-s was measured using first-void morning urine. In premenopausal women, the urinary aMT6-s level was significantly positively associated with percent density after controlling for covariates (P for trend=0.02). There was no significant association between urinary aMT6-s level and the percent density in postmenopausal women. We found no evidence that the melatonin level is inversely associated with mammographic density.

Biological monitoring of uranium exposure in south central Virginia

In May 2004, two groundwater wells in Dinwiddie County, Virginia were found to have natural uranium levels either at or above the EPA recommended limit of 30 microg/l. As a result, a stop drinking water advisory was issued until a water treatment system could be installed to remove the uranium. In response to residents' concerns, and uncertainty of exposures, affected individuals were asked to participate in a voluntary epidemiological investigation of uranium uptake and 1-year uranium retention study. This study had two primary objectives: quantification of the uranium load on the participants, as expressed by their urine uranium concentration, and retention after 1 year of no exposure. A first-morning void urine specimen, along with survey information, was collected from 156 participants in May 2004, with a second collection occurring 12 months later of 91 participants. The samples were analyzed for uranium by ICP/MS, pH, creatinine by the Jaffe method, and RBP by LIA after both collections. A reduction of one order of magnitude for the geometric mean urine uranium concentration was observed, from 0.100 microg/g creatinine to 0.011 microg/g creatinine in 1 year. Comparatively, NHANES has reported that the geometric mean for all participants, ages 6 years and older, is 0.008 microg/g creatinine, with the 95th percentile being 0.040 microg/g creatinine. None of the second round specimens showed a urine uranium concentration higher than baseline for an individual.

Variation in Nuclear DNA Concentrations During Urination*

This study examined the cellular origin and concentration of nuclear DNA in human urine. Ten subjects provided two entire, first-morning voids: one as a single specimen and one as a consecutive series of samples. The serial samples were centrifuged, organically extracted, and quantified by slot-blot analysis. Total DNA concentrations ranged from 0.02 to 21.3 ng/mL for the males and 25.0 to 96.9 ng/mL for the females. The female samples were found to contain numerous vaginal epithelial cells. DNA was detected in all of the serial samples of nine subjects; however, the DNA concentrations varied considerably. With six subjects, the DNA concentration of the first serial sample was at least three times greater than that of the entire void. DNA was only detected in the first 21% of the void from one male subject. The results of this study have implications for the collection of urine samples.

Prevalence of Daidzein-Metabolizing Phenotypes Differs between Caucasian and Korean American Women and Girls

Interindividual differences in metabolism of the soy isoflavone, daidzein, to equol and O-desmethylangolensin (ODMA) by human gut bacteria, have been associated with altered risk of cancer and other chronic diseases, according to some studies. Differences have been reported in the prevalence of the equol-producer phenotype among populations, with a higher prevalence in soy-consuming Asian populations than in Western populations. To date, prevalence of the daidzein-metabolizing phenotypes in Asians, compared with Caucasians, has not been evaluated in the context of a standardized phenotyping method. We assessed the prevalence of equol- and ODMA-producer phenotypes in 91 Korean American (KA) women and girls living in the Seattle, Washington area and compared this with previous similarly collected prevalence data in Caucasian American (CA) women and girls (n = 222). We also compared the dietary habits of the 2 groups. Isoflavonoid concentrations in first-void morning urines, collected after a 3-d soy challenge, were used to establish equol-, and ODMA-producer phenotypes (>44 microg/L). The prevalence of the equol-producer phenotype was higher (51 vs. 36%; P = 0.015) and the ODMA-producer phenotype was lower (84 vs. 92%, P = 0.03) in KA than in CA women and girls. KAs consumed approximately 3 times more soy foods than the CAs, but no significant associations were found between the consumption of soy foods and equol-producer phenotype. Our findings support the reports that, compared with Western populations, Asian populations have a higher equol-producer prevalence. The additional observation that the prevalence of the ODMA-producer phenotype is lower in KAs suggests that daidzein-metabolizing patterns in general may differ between KAs and CAs.

Serial crystalluria determination and the risk of recurrence in calcium stone formers

Urinary crystal precipitation is the necessary initial step in kidney stone formation. However, clinical relevance of crystalluria in the evaluation of stone formers is disputed. We serially determined crystalluria in first-voided morning urine samples, together with full 24-hour urine biochemistry, in 181 patients with idiopathic calcium nephrolithiasis who had formed at least one calcium-oxalate stone and were followed for at least 3 years under our care. All stone events which occurred prior to referral, then after entry in the study were recorded. As compared with 109 patients who had no evidence of stone recurrence during follow-up, the 72 patients who experienced >/= one recurrent stone event had a lower daily urine volume (1.74 +/- 0.06 vs. 2.26 +/- 0.05 L/day (mean +/- SEM) (P < 0.0001), higher urine calcium and oxalate concentrations, and daily calcium excretion, and they had more frequent crystalluria (68% vs. 23% of urine samples) (P < 0.0001). By multivariate Cox regression analysis, the hazard ratio for stone recurrence was 0.32 (95% CI 0.16-0.62) for 1 L increase in daily urine volume, 1.12 (1.09-1.24) for 1 mmol/L increase in urine calcium concentration, 1.24 (1.02-1.50) for 0.1 mmol/L increase in urine oxalate concentration and 27.8 (10.2-75.6) for crystalluria index. These data provide evidence that crystalluria, when repeatedly found in early morning urine samples, is highly predictive of the risk of stone recurrence in calcium stone formers. Serial search for crystalluria, a simple and cheap method, may be proposed as a useful tool for the monitoring of calcium stone formers, in addition to urine biochemistry.

Association of Vegetable Intake with Urinary 6-Sulfatoxymelatonin Level

Melatonin is present in plants consumed as vegetables; however, only a limited number of vegetables have been tested for melatonin. The antiproliferative, antioxidative, and immunostimulatory effects of melatonin have been reported from laboratory studies. The potential protective effects of vegetable against cancer and cardiovascular disease may be partially attributable to an increased melatonin intake from vegetables. As a first step to test this hypothesis, we evaluated whether vegetable intake is associated with an increased urinary melatonin in 289 community-dwelling Japanese women. Diet, including vegetable consumption, was assessed with a validated 169-item semiquantitative food-frequency questionnaire. Urinary 6-sulfatoxymelatonin (aMT6-s) was measured in the first-void morning urines. There was a significant positive association between vegetable intake and urinary aMT6-s levels. The mean urinary aMT6-s was 16% higher in women with the highest quartile of vegetable intake than it was in those with the lowest quartile of intake. This association may be explained by the melatonin contained in vegetables. However, data should be regarded as preliminary because it is impossible to estimate dietary melatonin intake from vegetables and or from the entire diet because of incomplete data for melatonin in plants.

Exposure of nursery school children and their parents and teachers to di-n-butylphthalate and butylbenzylphthalate

Some phthalates, among them di-n-butylphthalate (DnBP) and butylbenzylphthalate (BBzP), are known reproductive and developmental toxicants in animals and suspected endocrine disruptors in humans. Children are probably the most susceptible to these effects. To obtain an estimate of internal exposure to DnBP and BBzP we compared the excretion of their metabolites in the urine of nursery school children with that of their teachers and parents. We measured the urinary mono-ester metabolites of DnBP, mono-n-butylphthalate (MnBP), and BBzP, monobenzylphthalate (MBzP), in first-morning voids of 36 children (median age 4.7 years) and 19 adults (37.4 years). In all samples both metabolites were detected. Urinary MnBP concentrations (in microgrammes per litre) of the children and adults were 139 and 91.8 (median), respectively. MBzP concentrations were 22.1 microg/l and 12.7 microg/l (median), respectively. Concentrations in microgrammes per gramme creatinine for MnBP were 161 for the children and 91.8 for the adults (median). The maximum concentration found for children (2249 microg/g) was approximately 15-times higher than that for adults (149 microg/g). This maximum value for children was attributed to medication that contained DnBP. If this child was excluded, the maximum concentration was 517 microg/g. MBzP concentrations for children and adults were 37.0 microg/g and 9.8 microg/g (median), respectively. The maximum concentration found for children (193 microg/g) was approximately seven-times higher than that for adults (26.7 microg/g). Creatinine-adjusted concentrations were significantly higher for children for both MBzP and MnBP (P<0.0001). MnBP and MBzP exposures were found to correlate statistically significantly within the children's cohort (r=0.723, P<0.001). Within the children's cohort we found elevated MnBP exposure to be caused by augmented use of skin-care products (P<0.05). We have shown that the internal exposure to MnBP and MBzP in children is approximately two- to four-times higher than in adults. Correlation of internal MnBP with MBzP exposure points to common sources of exposure for both phthalates. DnBP exposure seems, at least in part, to be connected with the use of body/skin care products and certain medications.

Prevalence of renal impairment in type 2 diabetes: a primary care-based study

The purpose of the study was to determine the prevalence of renal impairment, and to assess its significance, within type 2 diabetics attending primary care settings. We performed a cross-sectional observation of patients with type 2 diabetes being the first 20 unselected cases seen by 178 family physicians from 16 of the 17 departments of Spain. Following variables were determined: serum creatinine, and glucose concentrations, HbA1c, proteinuria (dipstick test in a first-voided morning urine sample), blood pressure level, and associated cardiovascular disease. Data from 3583 type 2 diabetics were evaluated. Mean age was 64+/-10 years being 45% male. A serum creatinine = or >1.2 mg/dl was observed in 523 (15.5%) patients. Proteinuria was present in 794 (23.5%) cases, being = or >2+ in 215 (6.5%) subjects. Patients with a serum creatinine = or >1.2 mg/dl were older, showed a higher blood pressure level, and suffered from more cardiovascular disease (32.0 vs. 19.5%) than those with a serum creatinine < 1.2 mg/dl. In a multivariate analysis, this difference continued to be significant (OR 1.47; 95% CI 1.14 to 1.90; p=0.002). Patients with proteinuria showed a higher prevalence of cardiovascular disease (OR 1.83; 95% CI 1.47 to 2.27; p<0.0001) than those without proteinuria. This association was continuous with an increasing pattern from the no proteinuric to the = or >2+ proteinuric patients (p<0.001). There is a high prevalence of renal impairment, approximately of 25%, within type 2 diabetic patients seen at the primary care level. These patients have a higher cardiovascular disease prevalence than type 2 diabetics with no clinic renal disease.

Left ventricular mass assessed by electrocardiography and albumin excretion rate as a continuum in untreated essential hypertension.

To study an association between albumin excretion rate and left ventricular mass (LVM) determined by electrocardiogram (ECG)-based criteria, and with respect to ambulatory blood pressure, in patients with newly diagnosed and never-treated essential hypertension. We measured 24 h ambulatory blood pressure, albumin excretion rate and LVM in 74 patients with newly diagnosed (within the past year) and never-treated essential hypertension, who were admitted to the hypertension clinic (Ulleval University Hospital). Albumin excretion rate was evaluated by determination of the albumin:creatinine ratio in the first-voided morning urine sample. LVM was assessed by ECG using Cornell voltage-QRS duration product and Sokolow- Lyon criteria. Albumin excretion rate was significantly related to ambulatory blood pressure. There was a weak but significant negative correlation between the decrease in diastolic blood pressure during the night and the rate of excretion of albumin. The patients classified as dippers had a significantly lower albumin excretion rate compared with the non-dippers (1.36+/-0.83 compared with 1.68+/-0.87 mg/mmol; P = 0.03). LVM was found to be significantly related to ambulatory blood pressure. A weak relationship was observed between albumin excretion rate and LVM as evaluated by Cornell criterion (r = 0.23, P = 0.045). When only normoalbuminuric patients were included (i.e. four patients with microalbuminuria were excluded), the relationship was stronger (r = 0.36, P = 0.002). The present findings show a continuous relationship between albumin excretion rate, LVM and ambulatory blood pressure in newly diagnosed patients with essential hypertension, and suggest the occurrence of early effects on target organs (kidneys and heart). These associations were observed using easily applicable methods such as ECG monitoring and determination of the albumin:creatinine ratio in morning urine samples.

Limitations of urinary telomerase activity measurement in urothelial cancer

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10 6 cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37°C and 25°C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37°C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25°C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

The effect of diurnal and menstrual cyclicity and menopausal status on estrogen metabolites: Implications for disease-risk assessment

It has been proposed that the ratio of two estrogen metabolites, 2-hydroxyestrone (2-OHE 1) and 16α-hydroxyestrone (16α-OHE 1), may represent a marker to predict a woman’s risk for developing breast cancer and other estrogen-related disease. The present studies evaluated the potential confounders of type of sample, diurnal rhythm, menstrual cycle phase, and menopausal status on the ratio of 2/16α-OHE 1 using an urine-based monoclonal antibody enzyme immunoassay. Two initial studies to compare a 24-h urine collection with a first-morning void and to evaluate diurnal variation were performed. Subsequently, urine samples were collected every other day for 2 months from five premenopausal subjects to assess the impact of the menstrual cycle. Spot urine samples were then obtained from a total of 67 pre, peri-, early post-, and late post-menopausal women to assess the effect of menopausal status. No significant difference in the ratio of 2/16α-OHE 1 was found between a 24-h and first-morning void or over a 24-h period. No significant difference in the mean ratio of 2/16α-OHE 1 was found with the menstrual phase. Intra-individual variability was observed in the ratio of 2/16α-OHE 1, which was attributable to small fluctuations in the small denominator, 16α-OHE 1. No difference in the ratio of 2/16α-OHE 1 was observed in groups of women of different menopausal status. The data suggest that a first-morning void is representative of a 24-h collection and that the 2/16α-OHE 1 ratio is constant throughout a 24-h period. Moreover, menstrual phase and menopausal status do not appear to significantly influence the ratio of 2/16α-OHE 1.

Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass.

We hypothesized that lower ovarian and gonadotropin hormone concentrations would be associated with lower levels of peak bone mineral density (BMD) in apparently normally menstruating women who did not exercise intensively and did not report anorexia or bulimia. This hypothesis was evaluated using a case-with-control study design (n = 65) which was nested within a population-based longitudinal study of peak bone mass (Michigan Bone Health Study) with annual assessment in women aged 25-45 years (n = 582). Cases were 31 premenopausal women with BMD of the lumbar spine, femoral neck, and total body less than the 10th percentile of the distribution, where controls were 34 premenopausal women with BMD between the 50th and 75th percentile. BMD was measured by dual-energy X-ray absorptiometry. In addition to their annual measurement, these 65 participants collected first-voided morning urine specimens daily through two consecutive menstrual cycles. The urine from alternating days of this collection was analyzed for estrone-3-glucuronide (E1G), pregnanediol glucuronide (PdG), testosterone, and follicle-stimulating hormone by radioimmunoassay and these values adjusted for daily creatinine excretion levels. Additionally, analyses of daily urine specimens for luteinizing hormone (uLH) was undertaken to better characterize the possible uLH surge. Cases had significantly lower amounts of E1G (p = 0.009) and PdG (p = 0.002) than did controls, whether amounts were characterized by a mean value, the highest value, or the area under the curve, and after statistically controlling for body size. Further, when B-splines were used to fit lines to the E1G and PdG data across the menstrual cycle, the 95% confidence intervals (CIs) about the line for the controls consistently excluded and excluded and exceeded the 95% confidence bands for the cases in the time frame associated with the luteal phase in ovulatory cycles. Likewise, 95% CIs for the LH surge in controls exceeded the fitted line for cases around the time associates with the LH surge. The cases and controls were not different according to dietary intake (energy, protein, calcium), family history of osteoporosis, reproductive characteristics (parity, age at menarche, age of first pregnancy), follicular phase serum hormone levels, calciotropic hormone levels, or by evidence of perimenopause. We conclude that these healthy, menstruating women with BMD at the lowest 10th percentile from a population-based study had significantly lower urinary sex steroid hormone levels during the luteal phase of menstrual cycles as compared with hormone levels in premenopausal women with BMD between the 50th and 75th percentile of the same population-based study, even after considering the role of body size. These data suggest that subclinical decreases in circulating gonadal steroids may impair the attainment and/or maintenance of bone mass in otherwise reproductively normal women.

The Relationship of Cortisol Levels to Social Environment and Reproductive Functioning in Female Cotton-Top Tamarins, Saguinus oedipus

Cortisol levels were examined in 17 cotton-top tamarin monkeys (Saguinus oedipus) to determine: (1) if first-morning void urine sampling could be used as a noninvasive method for monitoring cortisol excretion. (2) if capture and bleeding were associated with changes in urinary cortisol levels, (3) the relative cortisol levels in reproductively active and reproductively suppressed females, and (4) the relationship between cortisol levels and changes in social condition in cotton-up tamarins. Mean urinary cortisol levels during ovarian cycling did not differ between captured and bled females and undisturbed females. Mean cortisol levels were significantly lower in females who were housed in their natal groups and reproductively suppressed than in the same females when they were removed and placed adjacent to a novel male and ovarian cycling began. For all females, mean cortisol levels were higher during the periovulatory period than during the nonperiovulatory period of the ovulatory cycle, with mean cortisol levels higher in newly cycling females than in long-term cycling females. No differences were found in mean cortisol levels between long-term cycling females and prepubertal females and postpubertal natal females. Cortisol levels from long-term cycling females were much lower than during the first two to three ovarian cycles occurring in newly cycling female tamarins, during the last part of pregnancy, and during the first 6 weeks postpartum. These data provide evidence that (1) suppression of ovarian cycling in postpubertal females remaining in their natal family is not associated with stress-induced changes in cortisol levels. (2) cortisol levels are not strictly tied to reproductive condition, and (3) social change may be reflected in cortisol elevation.

Airborne arsenic and urinary excretion of metabolites of inorganic arsenic among smelter workers

The relationship between airborne concentrations of arsenic and the urinary excretion of inorganic arsenic metabolites (inorganic arsenic + methylarsonic acid + dimethylarsinic acid) have been studied among smelter workers exposed to arsenic trioxide. The urinary concentrations of arsenic metabolites were found to increase steadily during the first day of the working week (after 2-3 d off from work), whereafter they reached a steady state. The concentration in the late evening after a day of exposure was very similar to that in the early morning after. Both were well correlated to the total daily excretion. In the second part of the study, comprising 18 subjects, the first-void morning urine of each participant was collected for 2 to 3 d during the steady-state phase. Total concentration of arsenic in the breathing zones was measured by personal air samplers. Airborne arsenic (8-h values) varied between 1 and 194 micrograms As/m3, and urinary arsenic between 16 and 328 micrograms As/g creatinine. With the urinary arsenic concentrations (mean values of 2-3 d for each subject) plotted against the corresponding airborne arsenic concentrations, the best fit was obtained by a power curve with the equation y = 17 X X0.56. However, four of the participants were found to excrete far more (105-260%) arsenic in the urine than possibly could have been inhaled, most likely due to oral intake of arsenic via contaminated hands, cigarettes or snuff. If these four were excluded, the best fit was obtained by a straight regression line with the slope 2.0 and the intercept 29 micrograms As/g creatinine (coefficient of correlation 0.92; P less than 0.001).

Radioimmunoassay for free and conjugated urinary metanephrine.

We describe a convenient and specific radioimmunoassay for urinary metanephrine, in which we used an antiserum generated in rabbits by injecting with synephrine conjugated to bovine serum albumin as described by Grota and Brown [Endocrinology 98, 615 (1976)]. The antiserum is specific for metanephrine and epinephrine. Epinephrine cross reaction is minimized to 1% by adding 2 ng of epinephrine to each assay tube. Incubation time is 12 h. The sensitivity of the assay is 40 pg. Within-and between-assay coefficients of variation are 7.0 and 9.7%, respectively. We assayed first-voided morning urine and 24-h urine specimens from 15 normal subjects before and after acid hydrolysis, to assess total and free metanephrine concentrations. Mean (+/-SD) total and free urinary metanephrine excretion were 17 +/- 11 and 3.5 +/- 1.0 micrograms/24 h in these subjects; corresponding values for the morning specimens were 13.4 +/- 8.3 and 3.5 +/- 1.5 micrograms/liter, respectively.