Circulating plasma microRNAs as biomarkers for iron status in blood donors.

OBJECTIVES: To investigate whether microRNAs can serve as biomarkers for iron status in blood donors. BACKGROUND: Serum ferritin is a widely used biochemical test for detecting iron deficiency, but it has its limitations. Certain microRNAs (miRNAs) reportedly have a role in regulating iron homeostasis. Circulating miRNAs have been reported as potential biomarkers for various conditions but have not yet been studied in iron deficiency. METHODS: Participating blood donors were divided into two groups: high ferritin (HF) (>150 µg L-1 ) and low ferritin (LF) (<15 µg L-1 ). MiRNA analysis was performed by an miRNA profiling service (Exiqon) using commercial qPCR assays. The study had two phases: (i) a pilot study (20 participants) where 179 miRNAs were analysed and (ii) a confirmation study (50 participants) of 13 selected miRNAs. RESULTS: Mean serum ferritin was 13·8 µg L-1 in the LF arm compared to 231 µg L-1 in the HF group (P < 0·001). Hepcidin plasma levels were higher in the HF arm (P < 0·001), whereas soluble transferrin receptor 1 was higher in the LF group (P < 0·001). In the pilot study, samples did not separate according to study group on unsupervised analysis. When directly comparing HF vs LF groups, 17 miRNAs were differentially expressed (P < 0·05, t-test) but did not pass correction for multiple testing. The confirmation study of 13 selected miRNAs verified these findings as no miRNA was significantly different between the study groups. CONCLUSION: In this study, circulating plasma miRNAs did not emerge as promising biomarkers for iron status in healthy individuals. However, in the future, alternative detection methods such as next-generation sequencing might indicate miRNAs that correlate with iron stores.

Morphological determinants of signal carrier frequency in katydids (Orthoptera): a comparative analysis using biophysical evidence of wing vibration.

Male katydids produce mating calls by stridulation using specialized structures on the forewings. The right wing (RW) bears a scraper connected to a drum-like cell known as the mirror and a left wing (LW) that overlaps the RW and bears a serrated vein on the ventral side, the stridulatory file. Sound is generated with the scraper sweeping across the file, producing vibrations that are amplified by the mirror. Using this sound generator, katydids exploit a range of song carrier frequencies (CF) unsurpassed by any other insect group, with species singing as low as 600 Hz and others as high as 150 kHz. Sound generator size has been shown to scale negatively with CF, but such observations derive from studies based on few species, without phylogenetic control, and/or using only the RW mirror length. We carried out a phylogenetic comparative analysis involving 94 species of katydids to study the relationship between LW and RW components of the sound generator and the CF of the male's mating call, while taking into account body size and phylogenetic relationships. The results showed that CF negatively scaled with all morphological measures, but was most strongly related to components of the sound generation system (file, LW and RW mirrors). Interestingly, the LW mirror (reduced and nonfunctional) predicted CF more accurately than the RW mirror, and body size is not a reliable CF predictor. Mathematical models were verified on known species for predicting CF in species for which sound is unknown (e.g. fossils or museum specimens).

Symptoms and side effects in chronic non-cancer pain patients: clinical implications and development of new assessment tools.

OBJECTIVE: To improve a 41-item screening tool evaluated in our previous study by making it more simple and convenient to patients and at the same time maintain the level of information and the sensitivity. METHODS: In a prospective, two-period questionnaire study, patients suffering from chronic pain of non-cancer origin for more than 6 months, were asked to fill in two questionnaires: QSSE-41 or QSSE-33 and SF-36. The first part of the study (QSSE-41) included an age- and sex-matched control group. RESULTS: A total of 67 patients were included in QSSE-41 and 60 patients in QSSE-33. In QSSE-41, the mean number of symptoms reported by the patient group (12.3) was significantly higher than those reported by the controls (6.8) (P < 0.001). Out of the total number of symptoms, 40.3% were reported to be side effects caused by analgesics, and out of those 61.3% were reported as acceptable and 38.7% as unacceptable side effects. In the QSSE-33, the mean number of symptoms reported by the patient group was 13.6. Out of the total number of symptoms, 46.3% were reported to be side effects caused by analgesics, and out of those 56.4% were reported as acceptable and 43.6% as unacceptable side effects. CONCLUSIONS: This new and shorter screening tool QSSE-33 may substitute the original QSSE-41 and in clinical use, contribute substantially to a more comprehensive and detailed understanding of symptoms/side effects and may consequently lead to improved therapies.

Cloxacillin concentrations in serum, subcutaneous fat, and muscle in patients with chronic critical limb ischemia.

BACKGROUND: Patients suffering from critical limb ischemia (CLI) have poor wound healing in the ankle and foot areas. Secondary wound infections are frequent and often treated with prolonged courses of antibiotics. PURPOSE: This study set out to investigate to what extent the unbound fraction of 4 g of cloxacillin i.v. reaches its target organ in poorly vascularized tissues, i.e., the calf and foot of patients suffering from CLI. METHODS: Cloxacillin concentrations were measured by HPLC in serum and in microdialysis samples from skin and muscle of the lower part of the calf and as reference subcutaneously at the pectoral level in eight patients suffering from CLI (four males, four females, mean age 78 years, range 66-85 years) and in three healthy controls (two females, one male, mean age 67, range 66-68 years). RESULTS: In patients suffering from CLI, the tissue penetration of cloxacillin after a single 4 g dose was comparable to that of healthy controls, despite impaired blood circulation. CONCLUSIONS: The reduced blood flow in the peripheral vessels of the CLI patients presented here apparently is not the rate-limiting factor for delivery or tissue penetration of cloxacillin.

Association of HLA-DRB1*01 with Dupuytren's disease.

OBJECTIVES: To explore the human leucocyte antigen (HLA)-DRB1 allele frequency in Dupuytren's disease (DD). METHOD: HLA-DRB1 genotypes were analysed by sequence-specific primers (SSPs) in samples collected from 172 men participating in a nested case-control study on the clinical manifestations and progression of DD. Of those, 121 had signs of DD while 51 did not. Of the 121 men with DD, 49 had contracted fingers or had been operated on, while 72 had nodules or fibrous cords in the palms. Odds ratios (ORs) and 95% confidence interval (CIs) were used to evaluate the results. RESULTS: The HLA-DRB1*01 allele was observed in 26 of the 121 affected men (23.7%) but in only four of the controls (7.8%) (OR 3.22, 95% CI 1.06-9.75). The HLA-DRB1*01 allele frequency in those affected was 11%, while in the control group it was 4% (OR 3.07, 95% CI 1.05-9.03). CONCLUSIONS: This observation indicates a possible association of HLA-DRB1*01 with DD, but further studies are needed for confirmation.

Symptoms and side effects in chronic non-cancer pain: patient report vs. systematic assessment.

BACKGROUND: relieving distressing symptoms and managing the side effects of analgesics are essential in order to improve quality of life and functional capacity in chronic non-cancer pain patients. A quick, reliable and valid tool for assessing symptoms and side effects is needed in order to optimize treatment. We aimed to investigate the symptoms reported by chronic non-cancer pain patients after open-ended questioning vs. a systematic assessment using a list of symptoms, and to assess whether the patients could distinguish between the symptoms and the side effects induced by analgesics. METHODS: patients treated with either opioids and/or adjuvant analgesics were asked to report their symptoms spontaneously, followed by a 41-item investigator-developed symptom checklist. A control group also filled in the checklist. RESULTS: a total of 62 patients and 64 controls participated in the study. The numbers of symptoms reported by the patients (9.9 ± 5.9) were significantly higher than those reported by the controls (3.2 ± 3.9) (P<0.001). In the patient group, the number of spontaneously reported symptoms (1.3 ± 1.4) was significantly lower than the symptoms reported when using the symptom checklist (9.9 ± 5.9) (P<0.001). The six most frequently symptoms reported by the patients were: (1) Fatigue; (2) Memory deficits; (3) Dry mouth; (4) Concentration deficits; (5) Sweating; and (6) Weight gain. Out of the six most frequently reported symptoms, the share of side effects due to analgesics was: (1) Dry mouth (42%); (2) Sweating (34%); (3) Weight gain (29%); (4) Memory deficits (24%); (5) Fatigue (19%); and (6) Concentration deficits (19%). CONCLUSION: the number of symptoms reported using systematic assessment was eightfold higher than those reported voluntarily. Fatigue, cognitive dysfunction, dry mouth, sweating and weight gain were the most frequently reported. The patients reported the side effects of their analgesics to contribute substantially to the reported symptoms.

High Mortality from Major Vascular Trauma in Traffic Accidents: A Population-Based Study.

BACKGROUND AND AIMS: Injuries involving major arteries are an important cause of mortality and morbidity, most often from road traffic accidents. Our aim was to study the outcome of major vascular trauma from traffic accidents in an entire population, including patients who die at the scene and those who reach hospital alive. MATERIALS AND METHODS: This was a retrospective analysis of all patients who sustained major vascular trauma in traffic accidents in Iceland from 2000 to 2011. Patient demographics, mechanism, and location of vascular injury and treatment were registered. Injury scores were calculated and overall survival estimated. RESULTS: There were 62 individuals (mean age 44 years, 79% males) with 95 major vascular traumas, giving an incidence of 1.69/100,000 inhabitants (95% confidence interval: 1.27-2.21). A total of 33 died at the scene and 8 during transportation to hospital but 21 (34%) reached hospital alive. Most patients who succumbed had thoracic major vascular traumas (76%) or abdominal major vascular traumas (23%). Mean new injury severity score for the 21 admitted patients was 44. A total of 18 were operated with vascular repair, 3 with endovascular stent graft insertion. The mean hospital stay for discharged patients was 34 days. Altogether, 15 of the 62 patients (24%) survived to discharge from hospital, with a 5-year survival of 86% for discharged patients. CONCLUSION: Every other patient with major vascular trauma following traffic accidents died at the scene and a further 13% died during transportation to hospital, most of whom sustained major vascular trauma to the thoracic aorta. However, one-third of the patients reached hospital alive and 71% of them survived to discharge, with excellent long-term survival.

Rare and Common Variants Conferring Risk of Tooth Agenesis.

We present association results from a large genome-wide association study of tooth agenesis (TA) as well as selective TA, including 1,944 subjects with congenitally missing teeth, excluding third molars, and 338,554 controls, all of European ancestry. We also tested the association of previously identified risk variants, for timing of tooth eruption and orofacial clefts, with TA. We report associations between TA and 9 novel risk variants. Five of these variants associate with selective TA, including a variant conferring risk of orofacial clefts. These results contribute to a deeper understanding of the genetic architecture of tooth development and disease. The few variants previously associated with TA were uncovered through candidate gene studies guided by mouse knockouts. Knowing the etiology and clinical features of TA is important for planning oral rehabilitation that often involves an interdisciplinary approach.

New nucleophilic catalysts for bright and fast peroxyoxalate chemiluminescence

Miniaturized detection applications based on chemiluminescence require fast reaction kinetics for optimum performance. In this work, high-intensity light from the analytically useful peroxyoxalate chemiluminescence reaction has been generated at high rates by employing both single-component and dual-component nucleophilic catalysis. 4-(Dimethylamine)pyridine and its derivatives were superior to all other bases in terms of reaction speed and intensity of the generated light and outshone imidazole, which hitherto has been considered as the best catalyst. The light intensity was related to the difference in pKa between the 4-aminopyridine catalyst and the leaving group of the reagent, and the optimum delta pKa was found to be close to 0. Similarly, high light intensities were obtained when mixtures of the imidazole analogue 1,2,4-triazole and the strong, nonnucleophilic base 1,2,2,6,6-pentamethylpiperidine acted as catalysts. The mechanism behind this was concluded to be a "base-induced nucleophilic catalysis", where the ancillary strong base assisted the production of the highly nucleophilic 1,2,4-triazolate anion, which as the actual catalyst then participated in the formation of a more reactive transient reagent. All the investigated catalysts reduced the light yield of the reaction due to base-catalyzed breakdown reactions of the reagents and/or intermediates. The intensity peak maximums of these bright and fast reactions typically appeared after less than 10 ms, whereafter the light decayed to darkness within a few seconds. These reaction characteristics are especially advantageous for sensitive detection applications where the observation volumes and times are limited, e.g., peaks emerging from a capillary-based separation process.

Pharmacokinetics and pharmacodynamics of melagatran, a novel synthetic LMW thrombin inhibitor, in patients with acute DVT.

Forty-eight patients with acute proximal deep vein thrombosis (DVT) were randomised to intravenous infusions for 4 to 6 days with melagatran, a novel synthetic low molecular weight thrombin inhibitor, or unfractionated heparin adjusted by the activated partial thromboplastin time (APTT). The aim of the study was to investigate the pharmacokinetics, pharmacodynamics and the safety of melagatran therapy at three different doses. Steady-state plasma concentrations were rapidly achieved and maintained throughout the infusion period. The mean plasma concentrations in the low, medium and high dose groups were 0.17, 0.31 and 0.53 micromol/l, respectively. The prolongation of APTT was stable during the melagatran infusions and correlated to the plasma concentration. Phlebographically verified regression of thrombus size measured as decrease in Marder score was seen after 4 to 6 days in 8 of 12 patients, 6 of 12 patients and 5 of 11 patients in the low, medium and high dose groups of melagatran and in 5 of the heparin-treated patients. In the low dose group with melagatran, thrombus extension was seen in one patient. At the dose levels studied, melagatran was well tolerated with no clinically significant bleeding problems, suggesting that melagatran could safely be given to patients suffering from DVT.

Epidemiology of Dupuytren's disease: clinical, serological, and social assessment. The Reykjavik Study.

Dupuytren's disease or palmar fibromatosis is a common disabling hand disorder, mainly confined to Caucasians of northwestern European origin. The prevalence of Dupuytren's disease and possible risk factors related to the disease were evaluated in a random sample of 1297 males and 868 females, aged 46 to 74 years. Blood samples were collected and biochemical parameters were evaluated. The possible relation between the disease and clinical, social, and biochemical parameters were estimated with age-adjusted univariate logistic regression analysis. Altogether 19.2% of the males and 4.4% of the female participants had clinical signs of Dupuytren's disease. The prevalence increased with age, from 7.2% among males in the age group 45-49 years up to 39.5% in those 70-74 years old. The more severe form of the disease, finger contractures, was found in 5.0% of the men and 1.4% had required operation, while this was rarely seen among women. In men elevated fasting blood glucose (P < 0.04), low body weight, and body mass index were significantly correlated with the presence of the disease (P < 0.001). Dupuytren's disease was common among heavy smokers (P = 0.02) and those having manual labor as occupation (P = 0.018). These results show that Dupuytren's disease is common in the Icelandic population and occupation and lifestyle seem to be related to the disease.

The effect of preoperative methylprednisolone on pulmonary function and pain after lung operations.

Thirty-six patients undergoing elective thoracotomy with pulmonary resection with the use of combined epidural and general anesthesia were randomized into a double-blind study to receive a single intravenous preoperative dose of methylprednisolone 25 mg/kg body weight or a placebo (saline solution). Postoperative pain relief consisted of epidural morphine 4 mg and paracetamol 1 gm three times a day for 4 days. Postoperative pulmonary function (peak expiratory flow rate, forced expiratory volume in first second, forced vital capacity) was evaluated on days 1, 2, 3, 4, and 7 and after 1 month. The value obtained after 1 month served as the control value. Pain score at rest and during cough was evaluated after 4 and 8 hours and on days 1, 2, 3, and 4. Pulmonary function was reduced after operation to the same degree in the steroid and placebo group: 42% versus 41% for forced expiratory volume in first second and 38% versus 39% for forced vital capacity, compared with control values after 1 month. Pain score was reduced in the steroid group after 4 hours and on day 1 during rest and after 4 and 8 hours and on day 2 during cough, compared with results in the placebo group (p < 0.05). In the steroid group three patients underwent reoperation because of leakage through the chest wall incision. In conclusion, administration of a single preoperative dose of methylprednisolone did not affect the postoperative reduction in pulmonary function after thoracotomy despite attenuated pain response, and the results do not warrant steroid administration before lung operation.

Does smoking stimulate rheumatoid factor production in non-rheumatic individuals?

Smoking has been associated with increased incidence of rheumatoid arthritis (RA), joint damage and positive rheumatoid factor (RF). Here we report an analysis of the association between smoking and IgM, IgG and IgA RF in a cohort of non-rheumatic individuals participating in a prospective longitudinal study of the incidence and significance of elevated RF. From the initial cohort of nearly 14,000 randomly selected individuals aged 52-80 years, 109 RF-positive and 187 RF-negative non-rheumatic participants were recruited. All participants were tested for RF at least twice at an interval ranging from 4 to 13 years. Of the RF-negative participants 21.9% were active smokers compared to 34.1% of IgM RF-positive (p=0.035), 20.8% of IgG RF-positive (N.S.) and 34.4% of IgA RF-positive participants (p=0.047). Smoking was most prevalent (44.8%) amongst participants with elevation of both IgM and IgA RF (p=0.008), and smokers were also significantly more likely to have a persistent elevation of RF than non-smokers (p=0.024). These findings indicate that smoking may influence the immune system, leading to increased production of IgM and IgA RF.

MK-801 selectively protects mouse arcuate neurons in vivo against glutamate toxicity.

The receptor mediating glutamate toxicity in vivo has not yet been identified. The effects of NMDA antagonist MK-801 and the AMPA antagonist NBQX were studied on glutamate toxicity in the arcuate nucleus of newborn and adult mice. Morphometric methods were used to determine the effects of antagonists on glutamate toxicity. In the developing arcuate, MK-801 abolished glutamate neurotoxicity whereas NBQX has no effect. MK-801, but not NBQX, afforded significant but not complete protection in mature arcuate neurons. The residual toxicity could not be blocked by co-administration of NBQX which paradoxically partially inhibited the protective effect of MK-801. The results show that throughout development, glutamate neurotoxicity in the arcuate nucleus is predominantly mediated by NMDA receptors.

Neutrophil-dependent decrease in early wound margin strength.

We evaluated the effect of neutropenia or administration of a serine proteinase inhibitor on the early suture-holding capacity of intestinal anastomoses in rats. One group of rats was treated with antineutrophil serum, and another group received the soybean trypsin inhibitor. Controls received inactivated serum or saline. Anastomotic suture-holding capacity (breaking strength), myeloperoxidase activity, and collagen were measured 0 and 72 hours after surgery. Suture-holding capacity decreased by 70% in controls and 35% in soybean trypsin inhibitor-treated rats, but remained on level with immediate postoperative strength in neutropenic rats, where low myeloperoxidase levels reflected effective wound margin neutropenia. Collagen content and solubility were similar in all groups. These findings indicate that reduction in early wound margin strength is neutrophil dependent, and that neutrophil serine proteinases are important mediators in that process.

Effects of Predator-prey Body Size Ratios on the Stability of Food Chains.

The effects of predator-prey body size ratios on the resilience and probability of stability in linear Lotka-Volterra food chains have been analysed. The prey per capita interaction strengths of the model is assumed to be negatively correlated to the relative size difference between a predator and its prey. The relationship between prey interaction strength and predator-prey body size ratios is motivated by energetical arguments. Analytical results show that, given this assumption (on prey interaction strengths) and if average (relative) size differences between predators and their prey decrease with the trophic position of the consumer (as found in a large number of "real food webs") the probability of local stability in model food chains is increased (when compared to model chains with a constant predator-prey body size ratio). Numerical simulations show that in most cases, the effect on the probability of stability is accompanied by an increase in resilience. For example, as model food chain length is increased from two to three trophic levels in one simulation, the return time increases by more than two orders of magnitude with a constant predator-prey body mass ratio while chains longer than four are not feasible. With a decreasing predator-prey body mass ratio on the other hand, the return time does not increase as rapidly and feasible equilibria exist for longer chains. The relationship between resilience and food chain length is, in this model, affected by the relationship between the predator-prey body mass ratio and the trophic position of the predator, that is, how fast this ratio decreases with increasing trophic height. The effect of body mass on consumer mortality rates, and subsequently on the probability of stability and resilience is also analysed. Decreasing mortality rates with increasing body size does not change the results qualitatively, it only increases the probability that an equilibrium is feasible.Copyright 1998 Academic Press

Effects of electrical muscle stimulation combined with voluntary contractions after knee ligament surgery.

The aim of the present study is to compare the effect of electrical muscle stimulation combined with voluntary muscle contractions with a program only with voluntary muscle contractions during immobilization in casts after anterior cruciate ligament surgery. Twenty-three patients, 7 women and 16 men with a mean age of 28 yr, were randomized into two groups: an experimental group (13 patients) and a control group (10 patients). Post-operatively, the patients were immobilized for 3 wk in a full leg cast with the knee flexed at an angle of 20 degrees to 30 degrees and then in a knee cast for another 3 wk. All patients had a standard program with quadriceps muscle contractions. In addition, the experimental group received electrical stimulation of the quadriceps muscle 4 X 10 min, 3 times.wk-1, at a frequency of 30 Hz. During each stimulation, the patients were requested to contract the quadriceps muscle voluntarily as well. When pre-operative measurements were compared with those at the end of the immobilization period (6 wk after the operation), a significantly larger reduction in the knee extension isometric muscle strength was found for the control group than for the experimental group. In comparisons of the data of the male subjects only, this difference was still seen to be significant. The cross-sectional area of the quadriceps muscle measured with computed tomography was significantly less reduced during the immobilization period in the experimental group than in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Body size and food web structure: testing the equiprobability assumption of the cascade model.

The cascade model successfuly predicts many patterns in reported food webs. A key assumption of this model is the existence of a predetermined trophic hierarchy; prey are always lower in the hierarchy than their predators. At least three studies have suggested that, in animal food webs, this hierarchy can be explained to a large extent by body size relationships. A second assumption of the standard cascade model is that trophic links not prohibited by the hierarchy occur with equal probability. Using nonparametric contingency table analyses, we tested this "equiprobability hypothesis" in 16 published animal food webs for which the adult body masses of the species had been estimated. We found that when the hierarchy was based on body size, the equiprobability hypothesis was rejected in favor of an alternative, "predator-dominance" hypothesis wherein the probability of a trophic link varies with the identity of the predator. Another alternative to equiprobabilty is that the probability of a trophic link depends upon the ratio of the body sizes of the two species. Using nonparametric regression and liklihood ratio tests, we show that a size-ratio based model represents a significant improvement over the cascade model. These results suggest that models with heterogeneous predation probabilities will fit food web data better than the homogeneous cascade model. They also suggest a new way to bridge the gap between static and dynamic food web models.

Augmentation with the longitudinal patellar retinaculum in the repair of an anterior cruciate ligament rupture.

Most authors advocate repair of the acutely ruptured ACL by augmentation or some form of primary reconstruction. Strong autogenous tissues like the patellar and semitendinosus tendons and the iliotibial band are often used when augmentation is the surgical choice. This paper describes a surgical procedure using a strip of the longitudinal patellar retinaculum as an augmentation of the repaired ACL. The retinaculum adds stability and strength to the repaired ACL during the healing period. Moreover, the retinaculum may enhance the healing process of the ruptured ACL by revascularization. It may also increase the stimulation of synovial tissue proliferation over the repaired ACL. Twenty-eight patients have been clinically and objectively evaluated at a mean follow-up time of 78 months (range, 63 to 94 months). The patients' own evaluation of their knee function was excellent or good in 86% (24 patients). Eighty-two percent (23 patients) could return to their preinjury activities at the same intensity level as before injury, whereas at an intermediate followup at 18 months only 64% (18 patients) had done so. Anterior tibial displacement was objectively evaluated at both followups by means of the anterior drawer test, with 20 degrees to 30 degrees and 90 degrees of knee flexion, in a testing device. The operated knee usually showed a tendency to a slightly increased anterior displacement when compared with the uninjured knee. The repair of a ruptured ACL with an augmentation using the longitudinal patellar retinaculum has been shown to give good results after more than 5 years.

Individual mercury exposure of chloralkali workers and its relation to blood and urinary mercury levels.

On two occasions, chloralkali workers were investigated with regard to personal air mercury exposure, blood mercury and urinary mercury. The first investigation (13 workers, 2 weeks) was made at an exposure above the threshold limit value (64 microgram/m3, range 36--112), the second (16 workers, 8 weeks) at a lower exposure (23 microgram/m3, range 15--43). At the higher level of exposure, good correlations were found between air exposure and blood or urinary mercury for the group, but not for individuals. At the lower level, the correlations were less pronounced for the group. For individuals, the best correlation was found between mean air exposure during one week and blood mercury about half a week later. Other individuals, mainly the least exposed, showed no such correlation. Corresponding correlations were not found for urinary mercury. The urinary excretion rate was determined only for the last few hours of the workday, but the results agree with earlier investigations of 24-h excretion on a group basis. The threshold limit value for mercury in air (50 microgram/m3) corresponds to 150--175 nmol Hg/1 blood (= 30--35 microgram/1) for the group, with large individual variation.