Development of a high-throughput screening analysis for 288 drugs and poisons in human blood using Orbitrap technology with gas chromatography-high resolution accurate mass spectrometry.

The screening analysis for drugs and poisons always symbolizes the capabilities of a forensic laboratory. Due to the rapid emergence of new compounds in clinical and forensic intoxication cases, sensitive and specific methods are necessary for the screening of wide range of target compounds. A novel high-throughput screening method has been developed for the toxicological analysis of 288 drugs and poisons in human blood using Orbitrap technology with gas chromatography-high resolution mass spectrometry (GC-HRMS). This method allows for the fast detection and identification of high-throughput forensically important drugs and poisons, e.g., drugs of abuse (cocaine, amphetamines, synthetic cannabinoids, opiates, hallucinogen), sedative-hypnotics, antidepressants, non-steroidal anti-inflammatory drugs, pesticides (acaricides, fungicides, insecticides, nematicides), and cardiovascular agents in one single GC-Q Exactive run. After a simple extraction with ethyl ether and buffer, following centrifugation, the supernatant was injected into the system. For detection, spiked blood samples were analyzed by Orbitrap-GC-HRMS using an electrospray ionization in full scan mode with a scan range from 40 to 650 (m/z). The identification of drugs and poisons in the samples was carried out by searching the accurate molecular mass of characteristic fragment ions, ion rations and retention time (RT) against the in-house library that we developed with 70 ev electron energy. The limit of detection (LOD) for most compounds (249 in a total of 288 compounds) was below 100 ng/mL. For selectivity, no substances have been identified in drug-free blood samples from six different sources, and the method was suitable for the recovery and the carryover. The coefficient of variation (CV) of the RTs was below 0.99% in all reproducibility experiments. Mass accuracy was always better than 3 ppm, corresponding to a maximum mass error of 1.04 millimass units (mmu). The developed method was applied to 136 real samples from forensic cases, demonstrating its suitability for the sensitive and fast screening of high-throughput drugs in human blood samples.

Ion Abundance Ratios of Qualitative Analysis by GC-MS of 4 Common Drugs （Poisons.

ABSTRACT: Objective To investigate the maximum allowable deviation of ion abundance ratios of characteristic fragment ions in common drugs （poisons） in blood by gas chromatography-mass spectrometry （GC-MS） method. Methods Four common drugs （poisons） （dichlorvos, phorate, diazepam and estazolam） were detected by GC-MS full scan mode after liquid-liquid extraction in two laboratories and under three chromatographic conditions. The deviations of ion abundance ratios of the four common drugs （poisons） in marked blood samples with concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 µg/mL were analyzed. At the same time, the false negative rates of ion abundance ratios were analyzed when the mass concentration was limit of detection （LOD）, 2LOD, limit of quantitation （LOQ） and 2LOQ, and the false positive rates of ion abundance ratios were analyzed with blank blood samples. Results Under the two laboratories, four common drugs （poisons） and three kinds of chromatography conditions, the differences in deviations of the ion abundance ratios of marked blood samples were not statistically significant （P>0.05）. More than 95% of the absolute deviations of the ion abundance ratios of the marked blood samples were within the range of ±10%, and more than 95% of the relative deviations were within the range of ±25%. In cases of low concentration （concentration less than 2LOQ） or low signal to noise ratio （3-15）, the false negative rate was less than 5% and the false positive rate was 0% when the relative deviation was greater than 50%. Conclusion The absolute deviations of ion abundance ratios of four common drugs （poisons） in marked blood samples are advised to have a determination range within ±10%, and the determination range of relative deviations within ±25%.

Ion Abundance Ratios of Qualitative Analysis by GC-MS of 4 Common Drugs （Poisons / 法医学杂志

Objective To investigate the maximum allowable deviation of ion abundance ratios of characteristic fragment ions in common drugs （poisons） in blood by gas chromatography-mass spectrometry （GC-MS） method. Methods Four common drugs （poisons） （dichlorvos, phorate, diazepam and estazolam） were detected by GC-MS full scan mode after liquid-liquid extraction in two laboratories and under three chromatographic conditions. The deviations of ion abundance ratios of the four common drugs （poisons） in marked blood samples with concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 μg/mL were analyzed. At the same time, the false negative rates of ion abundance ratios were analyzed when the mass concentration was limit of detection （LOD）, 2LOD, limit of quantitation （LOQ） and 2LOQ, and the false positive rates of ion abundance ratios were analyzed with blank blood samples. Results Under the two laboratories, four common drugs （poisons） and three kinds of chromatography conditions, the differences in deviations of the ion abundance ratios of marked blood samples were not statistically significant （P>0.05）. More than 95% of the absolute deviations of the ion abundance ratios of the marked blood samples were within the range of ±10%, and more than 95% of the relative deviations were within the range of ±25%. In cases of low concentration （concentration less than 2LOQ） or low signal to noise ratio （3-15）, the false negative rate was less than 5% and the false positive rate was 0% when the relative deviation was greater than 50%. Conclusion The absolute deviations of ion abundance ratios of four common drugs （poisons） in marked blood samples are advised to have a determination range within ±10%, and the determination range of relative deviations within ±25%.

Chronic aflatoxin exposure in children living in Bhaktapur, Nepal: Extension of the MAL-ED study.

Exposure to aflatoxin, a mycotoxin common in maize and groundnuts, has been associated with childhood stunting in sub-Saharan Africa. In an effort to further our understanding of growth impairment in relation to mycotoxins and other risk factors, biospecimens from a cohort of children enrolled in the Bhaktapur, Nepal MAL-ED study were assessed for aflatoxin exposure at 15, 24, and 36 months of age. Exposure was assessed through a well-established serum biomarker, the AFB1-lysine adduct. In this manuscript, the levels of aflatoxin exposure in the Nepal cohort were compared with those observed in aflatoxin studies, with child growth parameters as a health outcome. Results from this preliminary analysis demonstrated chronic aflatoxin exposure in children residing in Bhaktapur with a geometric mean of 3.62 pg AFB1-lysine/mg albumin. The range of exposure in this population is similar to those in African populations where associations with aflatoxin biomarkers and poor child growth have been observed. Future work will analyze the relationships between aflatoxin levels, growth, and other risk factors collected by the MAL-ED study.

Intervention trial with calcium montmorillonite clay in a south Texas population exposed to aflatoxin.

South Texas currently has the highest incidence of hepatocellular carcinoma (HCC) in the United States, a disease that disproportionately affects Latino populations in the region. Aflatoxin B1 (AFB1) is a potent liver carcinogen that has been shown to be present in a variety of foods in the United States, including corn and corn products. Importantly, it is a dietary risk factor contributing to a higher incidence of HCC in populations frequently consuming AFB1-contaminated diets. In a randomised double-blind placebo controlled trial, we evaluated the effects of a 3-month administration of ACCS100 (refined calcium montmorillonite clay) on serum AFB1-lysine adduct (AFB-Lys) level and serum biochemistry in 234 healthy men and women residing in Bexar and Medina counties, Texas. Participants recruited from 2012 to 2014 received either a placebo, 1.5 g or 3 g ACCS100 each day for 3 months, and no treatment during the fourth month. Adverse event rates were similar across treatment groups and no significant differences were observed for serum biochemistry and haematology parameters. Differences in levels of AFB-Lys at 1, 3 and 4 months were compared between placebo and active treatment groups. Although serum AFB-Lys levels were decreased by month 3 for both treatment groups, the low dose was the only treatment that was significant (p = 0.0005). In conclusion, the observed effect in the low-dose treatment group suggests that the use of ACCS100 may be a viable strategy to reduce dietary AFB1 bioavailability during aflatoxin outbreaks and potentially in populations chronically exposed to this carcinogen.

Indoxyl sulfate enhances IL-1ß-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1ß were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1ß expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1ß-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1ß-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1ß-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1ß-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

Occupational exposure to Aspergillus and aflatoxins among food-grain workers in India.

BACKGROUND: Aflatoxins are a metabolite of Aspergillus molds and are widespread in the natural environment. Workers who handle food grains are at increased risk of exposure to aflatoxins and subsequently certain respiratory conditions. In India, more than half of the employed population is engaged in some type of agricultural work, yet little known about the respiratory problems as a result of exposure to aflatoxins among workers who handle food grains in India. OBJECTIVES: The aim of this study was to determine the risk of occupational exposure to aflatoxins in food-grain workers compared to workers who are not occupationally exposed to food grains. METHODS: Bronchoalveolar lavage (BAL) and serum samples from 46 food-grain workers and 44 non-food-grain workers were analyzed for the presence of aflatoxins. Microscopy and culture of BAL samples were performed to detect Aspergillus species. RESULTS: Aflatoxins were detected in 32·6% of the food-grain workers and 9·1% of non food grain workers (P<0·01). A significant difference was also found in BAL culture for Aspergillus (P<0·01) between the two groups. About 47·8% of the food-grain workers and 11·4% of non-food-grain workers had chronic respiratory symptoms. CONCLUSION: Occupational exposure to aflatoxins in food-grain workers was found to be associated with the increased presence of respiratory symptoms.

Carcinogenic effects of aflatoxin B1 among wheat handlers.

BACKGROUND: Epidemiological studies have demonstrated that serum aflatoxin B1 (AFB1) is a hepatocarcinogenic mycotoxin and contributor to the high rate of hepatocellular carcinoma (HCC). The prevalence of liver cancer in Egypt is particularly worrisome. In a registry-based analysis of occupational risk for HCC, significant excesses were observed especially for grain mill workers. OBJECTIVE: The aim of this study was to assess the hepatic carcinogenicity of AFB1 in wheat handlers. METHODS: Serum AFB1/albumin (AFB1/Alb), alpha-fetoprotein (AFP), alpha-l-fucosidase (AFU), and arginase were estimated in exposed wheat handlers including millers and bakers. The control group was composed of non-occupationally exposed workers. RESULTS: AFB1/Alb and AFU were significantly higher among workers employed as bakers compared to mill workers and controls. Mill workers had higher levels of AFB1/Alb than the controls. AFB1/Alb, AFP, and AFU were all significantly higher and arginase was significantly lower among HCC cases compared to the other groups. There was a significant correlation between AFU and AFB1/Alb in bakers and between AFP and AFB1/Alb in HCC cases. Arginase was inversely correlated with AFB1/Alb in HCC cases. AFB1/Alb was significantly correlated with the duration of exposure in bakers. CONCLUSION: Wheat handlers exposed to Aspergillus flavus have a high risk of elevated serum AFB1/Alb levels and AFU.

Hemoperfusion for the treatment of poisoning: technology, determinants of poison clearance, and application in clinical practice.

Hemoperfusion is an extracorporeal treatment based on adsorption, historically reserved for the treatment of acute poisonings. Its use was popularized in the 1970s after several in vitro and animal experiments had demonstrated its efficacy, and was even preferred over hemodialysis in the management of overdosed patients. With the advent of new and more efficient dialytic modalities, hemoperfusion is now less frequently performed in the Western world. However, hemoperfusion still remains popular in developing countries. The present article reviews the technique of hemoperfusion, the factors influencing poison clearance through adsorption and its current applications.

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100µL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5µm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology.

Lead excretion in milk of accidentally exposed dairy cattle.

Lead (Pb) exposure in dairy cattle is associated with economic losses due to mortality and treatment costs, but with production animals there is also risk to the human food chain. The first objective of this study was to quantify the Pb concentration in milk from Pb-exposed cattle. The second objective was to correlate blood and milk Pb concentrations from individual cows. The third objective was long-term monitoring to determine the duration of milk contamination after exposure ceased. A dairy herd of more than 100 cows was accidentally exposed to Pb-contaminated feed. Milk and blood were collected for Pb analysis. Serial collection of milk samples continued for 2.5 years. The initial concentration of Pb in bulk tank milk was 0.0999 mg l⁻¹. The highest milk Pb concentration from an individual cow was 0.4657 mg l⁻¹ and the highest blood Pb concentration was 1.216 mg l⁻¹. One milk sample collected at the end of the study (day 922) contained 0.0117 mg Pb l⁻¹ of Pb. The calculated relationship between milk (y) and blood (x) Pb concentration was ln(y) = 3.4(x) - 2.21 (R² = 0.98).

Occupational exposure to aflatoxin B1 in swine production and possible contamination sources.

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Rapid drug extraction from human whole blood using a modified QuEChERS extraction method.

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of forensically important drugs and poisons (more than 90 compounds) in human whole blood. Because the QuEChERS method is commonly used for the analysis of pesticide residues in foods, we customized the QuEChERS method for forensic use. This extraction method consists essentially of two steps: (1) extraction/partitioning and (2) dispersive-solid phase extraction. In step 1, three-fold diluted blood was mixed with an internal standard (D5-diazepam for basic drugs or D5-phenobarbital for acidic drugs) solution, a QuEChERS pre-packed extraction kit (containing magnesium sulfate and sodium acetate) and a stainless steel bead, then partitioned into three layers by centrifugation. In step 2, the top layer (acetonitrile) was transferred into a centrifuge tube containing a dispersive-solid phase sorbent (containing primary secondary amine, end-capped octadecylsilane, and magnesium sulfate) and mixed for purification. After the centrifugation, supernatant was injected into LC-MS/MS. The QuEChERS method was applied in an autopsy case and we confirmed that this method can easily extract various types of drugs and metabolites from human whole blood. The combination of the modified QuEChERS method and LC-MS/MS could enable technicians inexperienced in forensic toxicological analysis to acquire reliable data quickly and easily.

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography-tandem mass spectroscopy and its application.

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 µm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.

[Bull's blood as an ancient poison]. / Bycza krew jako antyczna trucizna.

Article presents ancient tradition about poisoning with bull's blood and the modern attempts of its explanation. Greek and Roman literary sources are compared with the ancient medical texts.

[Quantitative determination of strychnine in blood and urine by gas chromatography with mass-selective detector].

A method for the quantitative determination of strychnine in biological fluids by gas chromatography--mass spectrometry is proposed. The preparation of samples for the analysis included extraction of strychnine from blood and urine with the use of AccuBond(II) EVIDEX cartridges for solid-phase extraction and SPEC MP3 disks respectively. The efficiency of extraction was estimated at 0.05 mg/l for blood and 0.02 mg/l for urine. The detection limit was 0.10 mg/l in blood and 0.05 mg/l in urine.

Association between birth outcomes and aflatoxin B1 biomarker blood levels in pregnant women in Kumasi, Ghana.

OBJECTIVE: To investigate the association between birth outcomes and blood levels of aflatoxin B(1) (AFB1)-lysine adduct in pregnant women in Kumasi, Ghana. METHOD: A cross-sectional study of 785 pregnant women attending antenatal clinic was conducted. Aflatoxin B(1) (AFB(1))-lysine adduct levels were determined by high performance liquid chromatography (HPLC) on blood taken after delivery. The birth outcomes considered were small for gestation age, low birthweight, preterm delivery and stillbirth. Participants were divided into quartiles based on the distribution of aflatoxin B(1)-lysine adducts in pg/mg albumin ('low': 2.67 to 4.97 to 11.34). Statistical analysis involved models that included socio-demographic variables and other potential confounders. RESULTS: The average AFB(1)-lysine adduct level in maternal serum was 10.9 +/- 19.00 pg/mg albumin (range = 0.44-268.73 pg/mg). After adjusting for socio-demographic variables and potential confounding factors, participants in the highest AFB(1)-lysine quartile with 'very high' AFB(1)-lysine level (>11.34 pg/mg) were more likely to have low birthweight babies (OR, 2.09; 95% CI, 1.19-3.68), and showed a trend of increasing risk for low birthweight (P(trend) = 0.007) compared to participants in the lowest quartile. CONCLUSION: This study adds to the growing body of evidence that aflatoxins may increase the risk of adverse birth outcomes. The findings have implications for targeted nutritional education of pregnant women in areas with high levels of aflatoxin contamination of foods.

Aflatoxin-albumin adducts and correlation with decreased serum levels of vitamins A and E in an adult Ghanaian population.

A study of aflatoxin (AF) exposure and the levels of vitamins A and E was carried out with a group of 507 Ghanaian participants. AFB(1)-albumin adducts (AFB-AA) were measured by radioimmunoassay and vitamins A and E were measured by high-performance liquid chromatography (HPLC). The average level of serum AFB-AA was 0.94 +/- 0.64 (range = 0.1-4.44) pmol mg(-1) albumin. Mean levels of vitamins A and E were 1.32 +/- 0.48 (range = 0.41-4.85) micromol l(-1) and 15.68 +/- 4.12 (range = 6.35-30.40) micromol l(-1), respectively. A significantly negative correlation was found between serum AFB-AA and vitamin A levels (r = -0.110, p = 0.013). An even stronger, significant negative, correlation was found between serum AFB-AA and vitamin E levels (r = -0.149, p < 0.001). Serum AFB-AA levels were statistically higher (median = 0.985 pmol mg(-1) albumin) in subjects who had low levels of both vitamins A and E as compared with the levels (median = 0.741 pmol mg(-1) albumin) subjects who had high vitamins A and E levels (p(trend) = 0.001). To verify these findings, blood samples were again collected from 165 of the 507 people 3 months after the initial collection. Significantly negative correlations were confirmed between levels of serum AFB-AA and both vitamins A (r = -0.232, p = 0.003) and E (r = -0.178, p = 0.023). Again, high serum AFB-AA concentrations (median = 1.578 pmol mg(-1) albumin) were found in subjects with low levels of vitamins A and E compared with the concentrations (median = 1.381 pmol mg(-1) albumin) in subjects with high levels of vitamins A and E (p(trend) = 0.002). These data show that AF exposure was associated with decreased levels of serum vitamins A and E in high-risk human populations, which may significantly influence the incidence of AF-related adverse health effects.

Ventilation in Pacific hagfish (Eptatretus stoutii) during exposure to acute hypoxia or hypercapnia.

A technique was developed to measure ventilation in unrestrained Pacific hagfish (Eptatretus stoutii) by inserting and fastening into the nostril a flexible tube fitted with an ultrasonic flow probe. This technique permitted the continuous measurement of ventilation (respiratory) frequency (fR), stroke volume and minute ventilation (.V(E)) in real time in fish exposed to acute hypoxia or hypercapnia. Exposing fish to acute hypoxia (final PW(O2)=21.0 +/- 3.4 mm Hg) caused hypoxaemia and a marked increase in .V(E) of 350+/-71 ml min(-1)kg(-1) (from 235 to 585 ml min(-1)kg(-1)) owing exclusively to an increase in fR of 44+/-7 min(-1) (from 19 to 63 min(-1)). Because O(2) consumption (approximately 0.4 mmol kg(-1)h(-1)) was unaltered during hypoxia, there was an associated marked increase in the ventilation convection requirement from 36.7 to 81.8l mmol(-1). Injecting the O(2) chemoreceptor stimulant NaCN into inspired water (external CN-) or pre-branchial blood (internal CN-) evoked ventilatory responses that were similar to those observed during hypoxia although of a lesser magnitude. With external CN(-), V (E) increased maximally by 146+/-46 ml min(-1)kg(-1) and fR increased by 20+/-2 min(-1). With internal CN-, the maximal increase in .V(E) was 93+/-30 ml min(-1)kg(-1) and fR increased maximally by 19+/-6 min(-1). Exposure to acute hypercapnia (final PwC=7.0+/-0.2 mmHg) caused an increase in V (E) of 169+/-60 ml min(-1)kg(-1). These results provide compelling evidence for chemoreceptor-mediated control of breathing in hagfish and suggest that ventilatory responses to environmental hypoxia and hypercapnia in the vertebrates arose in the myxine lineage.