Natural products show diverse mechanisms of action against Clostridium difficile.

AIMS: To investigate the mechanisms of action of natural products with bactericidal (cinnamon root powder, peppermint oil, trans-cinnamaldehyde, menthol and zingerone) or bacteriostatic (fresh garlic bulb extract, garlic clove powder, Leptospermum honey and allicin) activity against two Clostridium difficile strains. METHODS AND RESULTS: Bactericidal products significantly reduced intracellular ATP after 1 h (P ≤ 0·01), quantified using the BacTiter-Glo reagent, and damaged the cell membrane, shown by the leakage of both 260-nm-absorbing materials and protein, and the uptake of propidium iodide. Bacteriolysis was not observed, determined by measuring optical density of treated cell suspensions at 620-nm. The effect of three bacteriostatic products on protein synthesis was quantified using an Escherichia coli S30 extract system, with Leptospermum honey (16% w/v) showing significant inhibition (P < 0·01). Lastly, no products showed elevated minimum inhibitory concentrations against antimicrobial-resistant C. difficile, determined by broth microdilution. CONCLUSIONS: Cytoplasmic membrane damage was identified as a mechanism of action that may contribute to the activity of several natural products against C. difficile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the possible mechanisms of action of natural products against C. difficile, yet the efficacy in vivo to be determined.

In silico, in vitro and in vivo analysis of putative virulence factors identified in large clostridial toxin-negative, binary toxin- producing C. difficile strains.

The relevance of large clostridial toxin-negative, binary toxin-producing (A-B-CDT+) Clostridium difficile strains in human infection is still controversial. In this study, we investigated putative virulence traits that may contribute to the role of A-B-CDT+C. difficile strains in idiopathic diarrhea. Phenotypic assays were conducted on 148 strains of C. difficile comprising 10 different A-B-CDT+C. difficile ribotypes (RTs): 033, 238, 239, 288, 585, 586, QX143, QX444, QX521 and QX629. A subset of these isolates (n = 53) was whole-genome sequenced to identify genetic loci associated with virulence and survival. Motility studies showed that with the exception of RT 239 all RTs tested were non-motile. C. difficile RTs 033 and 288 had deletions in the F2 and F3 regions of their flagella operon while the F2 region was absent from strains of RTs 238, 585, 586, QX143, QX444, QX521 and QX629. The flagellin and flagella cap genes, fliC and fliD, respectively, involved in adherence and host colonization, were conserved in all strains, including reference strains. All A-B-CDT+C. difficile strains produced at least three extracellular enzymes (deoxyribonuclease, esterase and mucinase) indicating that these are important extracellular proteins. The toxicity of A-B-CDT+C. difficile strains in Vero cells was confirmed, however, pathogenicity was not demonstrated in a mouse model of disease. Despite successful colonization by most strains, there was no evidence of disease in mice. This study provides the first in-depth analysis of A-B-CDT+C. difficile strains and contributes to the current limited knowledge of these strains as a cause of C. difficile infection.

Clostridium difficile infection diagnosis in a paediatric population: comparison of methodologies.

The increasing incidence of Clostridium difficile infection (CDI) in paediatric hospitalised populations, combined with the emergence of hypervirulent strains, community-acquired CDI and the need for prompt treatment and infection control, makes the rapid, accurate diagnosis of CDI crucial. We validated commonly used C. difficile diagnostic tests in a paediatric hospital population. From October 2011 to January 2012, 150 consecutive stools were collected from 75 patients at a tertiary paediatric hospital in Perth, Western Australia. Stools were tested using: C. Diff Quik Chek Complete, Illumigene C. difficile, GeneOhm Cdiff, cycloserine cefoxitin fructose agar (CCFA) culture, and cell culture cytotoxin neutralisation assay (CCNA). The reference standard was growth on CCFA or Cdiff Chromagar and PCR on isolates to detect tcdA, tcdB, cdtA, and cdtB. Isolates were PCR ribotyped. The prevalence of CDI was high (43 % of patients). Quik Chek Complete glutamate dehydrogenase (GDH) demonstrated a low negative predictive value (NPV) (93 %). Both CCNA and Quik Chek Complete toxin A/B had poor sensitivity (33 % and 29 % respectively). Molecular methods both had 89 % sensitivity. Algorithms using GDH + Illumigene or GeneOhm reduced the sensitivity to 85 % and 83 % respectively. Ribotype UK014/20 predominated. GDH NPV and GeneOhm and Illumigene sensitivities were reduced compared with adult studies. Quik Chek Complete and CCNA cannot reliably detect toxigenic CDI. A GDH first algorithm showed reduced sensitivity. In a high prevalence paediatric population, molecular methods alone are recommended over the use of GDH algorithm or culture and CCNA, as they demonstrate the best test performance characteristics.

Clostridium difficile and One Health.

BACKGROUND: For over four decades, Clostridium difficile has been a significant enteric pathogen of humans. It is associated with the use of antimicrobials that generally disrupt the microbiota of the gastrointestinal tract. Previously, it was thought that C. difficile was primarily a hospital-acquired infection; however, with the emergence of community-associated cases, and whole-genome sequencing suggesting the majority of the hospital C. difficile infection (CDI) cases are genetically distinct from one another, there is compelling evidence that sources/reservoirs of C. difficile outside hospitals play a significant role in the transmission of CDI. OBJECTIVES: To review the 'One Health' aspects of CDI, focusing on how community sources/reservoirs might be acting as a conduit in the transfer of C. difficile between animals and humans. The importance of a One Health approach in managing CDI is discussed. SOURCES: A literature search was performed on PubMed and Web of Science for relevant papers published from 1 January 2000 to 10 July 2019. CONTENT: We present evidence that demonstrates transmission of C. difficile in hospitals from asymptomatic carriers to symptomatic CDI patients. The source of colonization is most probably community reservoirs, such as foods and the environment, where toxigenic C. difficile strains have frequently been isolated. With high-resolution genomic sequencing, the transmission of C. difficile between animals and humans can be demonstrated, despite a clear epidemiological link often being absent. The ways in which C. difficile from animals and humans can disseminate through foods and the environment are discussed, and an interconnected transmission pathway for C. difficile involving food animals, humans and the environment is presented. IMPLICATIONS: Clostridium difficile is a well-established pathogen of both humans and animals that contaminates foods and the environment. To manage CDI, a One Health approach with the collaboration of clinicians, veterinarians, environmentalists and policy-makers is paramount.

Contamination of Australian newborn calf carcasses at slaughter with Clostridium difficile.

In North America and Europe, reports of a genetic overlap between toxigenic strains of Clostridium difficile isolated from humans, livestock and retail meat suggest that food-borne transmission may be occurring. We investigated the prevalence, concentration and genetic diversity of C. difficile on the carcasses (n = 300) and in the faeces (n = 30) of neonatal veal calves at three abattoirs in Australia in 2013. Selective culture (both direct and enrichment) was performed, and all isolates were characterized by PCR for the toxin genes tcdA, tcdB and cdtA/B and by PCR ribotyping. Prevalence of C. difficile was 25.3% (76/300) on carcasses and 60.0% (18/30) in faeces. Multiple PCR ribotypes (RT) were detected, with four binary toxin-positive RTs accounting for 70.3% (71/101) of isolates; 127 (A(+), B(+), CDT(+), 32.7%), 288 (A(-), B(-), CDT(+), 28.7%), 033 (A(-), B(-), CDT(+), 6.9%) and 126 (A(+), B(+), CDT(+), 2.0%). Viable counts of a subset of samples revealed detectable numbers of C. difficile in 66.7% (10/15) of faecal samples (range 2.0 × 10(3) to 2.3 × 10(6) CFU/mL, median count 2.5 × 10(4) CFU/mL) and in 16.7% (25/150) of carcase samples (range 3 to 33 CFU/cm(2), median count 7 CFU/cm(2)). These data further confirm that Australian neonatal veal calf carcasses are contaminated with potentially significant strains of C. difficile at slaughter.

Effects of renin inhibition compared to angiotensin converting enzyme inhibition in conscious dogs with pacing-induced heart failure.

OBJECTIVE: To compare the effects of angiotensin converting enzyme inhibition (ACEI) (captopril 1 mg/kg i.v.) to direct renin inhibition (CP80794 3 mg/kg i.v.) on left ventricular and systemic hemodynamics and peripheral blood flows in advanced congestive heart failure (CHF). METHODS: Conscious chronically instrumented dogs (n = 14) were treated with captopril, 1 mg/kg, i.v., or CP80794, 3 mg/kg, i.v., before and after development of advanced CHF induced by 4-7 weeks of rapid ventricular pacing. After advanced CHF, comparisons between the inhibitors were made at equihypotensive doses. RESULTS: In advanced CHF, both agents caused comparable reductions in mean arterial pressure (MAP) (-22% from 79 +/- 4 mmHg) and comparable increases (P < 0.01) in cardiac output (CP80794, 1.4 +/- 0.3 to 1.8 +/- 0.1 l/min; captopril, 1.4 +/- 0.1 to 1.9 +/- 0.1 l/min). Neither agent had a significant effect on LV contractility. In contrast, CP80794 caused a greater (P < 0.05) increase in renal blood flow (66 +/- 6% from 64 +/- 5 ml/min) compared to captopril (33 +/- 4% from 66 +/- 7 ml/min). CONCLUSIONS: Renin inhibition with CP80794 and ACEI with captopril caused comparable hemodynamic effects in advanced CHF. However, CP80794 caused significantly greater increases in renal blood flow and suppressed renin activity to a greater degree than captopril.

Measurement of organ blood flow with coloured microspheres in the rat.

OBJECTIVE: The aim was to establish a method for measuring organ blood flow in rats using commercially available, coloured, dye extraction microspheres. METHODS: A mixture of radiolabelled and dye extraction microspheres was infused into rats at rest (basal) and during intravenous administration of either angiotensin II (0.5 microgram.kg-1.min-1) or isoprenaline [12.5 ng.(g0.74)-1.min-1]. Tissues were removed and placed in test tubes, counted for radioactivity, then digested with 2N sodium hydroxide. Within the same tube, microspheres were isolated using centrifugation and the dye was extracted with dimethylformamide. The dye was quantified by spectrophotometry. RESULTS: Recovery of microspheres averaged greater than 95% for all tissues studied; larger reagent volumes were required to achieve this level of recovery from white adipose tissue. Statistical analyses showed excellent correlations between blood flow values obtained by the dye extraction and radiolabelled microsphere techniques. Blood flow values obtained with the radioactive technique tended to be slightly higher. There were no differences in the results obtained with the two techniques when they were simultaneously used to measure changes in organ blood flow induced by angiotensin II or isoprenaline. CONCLUSIONS: The coloured, dye extraction microsphere technique accurately measured organ blood flow in rats. This technique is potentially useful for estimating blood flow in any animal, even if tissue sample size is limited.

Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart.

OBJECTIVE: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia.

OBJECTIVE: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on Ki values for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. METHODS: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols (n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. RESULTS: 5 nM CPA (52.2 +/- 3.1%), 30 nM IBMECA (49.7 +/- 3.8%) and preconditioning (55.3 +/- 2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7 +/- 1.0%; P < 0.05, ANOVA). When DPCPX (200 nM) was added prior to 5 nM CPA, protection was lost (31.8 +/- 0.9%), but when added prior to 30 nM IBMECA, muscles continued to be significantly protected (41.5 +/- 2.3%). CONCLUSIONS: In human atrium both A1 and A3 receptor stimulation appears to mimic ischaemic preconditioning. This may represent the first evidence for A3 receptor involvement in 'pharmacological' preconditioning of human myocardium.

Effects of a calcium channel blocker on cardiac output distribution in conscious hypertensive dogs.

The effects of nitrendipine, 8 micrograms/kg/minute, were evaluated in six conscious dogs through measurements of arterial pressure and blood flow in the ascending aorta (cardiac output), mesenteric, renal, and iliac arteries before and after induction of chronic perinephritic hypertension. Before hypertension was induced, nitrendipine reduced mean arterial pressure 19 +/- 2.3% (from 95 +/- 3.2 mm Hg), decreased total peripheral resistance (60 +/- 2.6%), and increased cardiac output (108 +/- 10.5%). These values returned to baseline within 15 to 30 minutes. Nitrendipine caused the greatest increase in blood flow in the iliac bed (98 +/- 9.9%), an intermediate increase in the mesenteric bed (37 +/- 3.7%), and the least increase in the renal bed (7 +/- 2.2%). Two to six weeks after induction of hypertension, administration of nitrendipine elicited significant (p less than 0.01) decreases in mean arterial pressure (32 +/- 2.5% from 151 +/- 4.8 mm Hg) and total peripheral resistance (67 +/- 1.3%) compared with its administration in normotensive dogs, while the increase in cardiac output was not significantly changed (111 +/- 10.9%). These changes in arterial pressure and vascular resistances also were prolonged (i.e., hemodynamics returned to baseline after 75-90 minutes). The increase in iliac (99 +/- 16.8%) and renal (9 +/- 6.1%) blood flows after nitrendipine administration in hypertensive dogs was similar to that found in the normotensive dogs, but mesenteric blood flow doubled (84 +/- 8.4%). Thus, in conscious, hypertensive dogs, nitrendipine administration appears to markedly decrease arterial pressure and total peripheral and regional resistances, which also require more time to return to baseline, but appears to increase blood flow by a greater amount only in the mesenteric bed.

Effects of renin inhibition in the conscious primate Macaca fascicularis.

Pro-His-Pro-Phe-His-Statine-Ile-Phe-NH2 (R-Pep-27), a potent renin inhibitory peptide, was infused into the conscious, sodium-depleted Macaca fascicularis at doses of 0, 0.1, 1, 4, 16, and 32 micrograms/kg/min for 10 minutes. At all doses greater than 0.1 microgram/kg/min, there was a parallel decrease in mean arterial pressure (MAP), plasma renin activity, and plasma angiotensin II (Ang II) concentration. On the other hand, assays with monoclonal antibodies specific for total renin and active renin demonstrated that the peptide's inhibition of circulating active renin stimulated the release of both. The maximal effective R-Pep-27 dose was approximately 16 micrograms/kg/min, which reduced MAP by an average of 15.8 +/- 1.4 mm Hg (n = 14) and plasma renin activity and plasma Ang II concentration to 3% (n = 9) and 15% (n = 5), respectively, of the pretreatment values. At 0.1 microgram/kg/min, there was no significant decrease in MAP; however, measurement of plasma renin activity showed an average decrease in activity of 42% (n = 3). No significant change in the heart rate was observed at all the doses studied. For comparison, intravenous captopril (400 micrograms/kg bolus) was administered after the MAP of the monkeys had recovered from the peptide experiments, and it reduced MAP by 25.1 +/- 2.4 mm Hg (n = 10) without significantly changing plasma renin activity. As anticipated, injection of angiotensin I (80-160 ng/kg bolus) into sodium-depleted monkeys during peptide infusion caused a transient rise in MAP of 14.8 +/- 5.4 mm Hg (n = 4) above the mean pretreatment value.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoconstrictor action of angiotensin I-convertase and the synthetic substrate (Pro11,D-Ala12)-angiotensin I.

A chymase (also referred to as angiotensin I-convertase) specific for the conversion of angiotensin (Ang) I to Ang II has been identified in human heart. This serine protease is also present in dog and marmoset vasculature. We examined the vasoconstrictor effects of Ang II putatively generated from an angiotensin-converting enzyme (ACE)-resistant convertase synthetic substrate (SUB) in vivo and in vitro. In marmosets, SUB (7 to 700 micrograms/kg i.v.) or Ang I (0.1 to 30 micrograms/kg) caused similar dose-dependent increases in mean arterial pressure (10 to 100 mm Hg) and decreases in heart rate. Pressor effects of SUB were slightly attenuated at low (but not high) doses by captopril (CAP, 1 mg/kg i.v.) and blocked by losartan (5 mg/kg i.v.); in contrast Ang I pressor effects were substantially blocked by both. In isolated canine superior mesenteric artery, Ang I-induced contraction was eliminated by losartan and reduced but not eliminated by 10 mumol/L CAP. When combined with the serine protease inhibitor chymostatin, CAP eliminated Ang I-induced contraction, but chymostatin alone had no effect. SUB-induced contraction was not blocked by CAP but was equally blocked by chymostatin (25 mumol/L) alone or by the combination of CAP (10 mumol/L) and chymostatin (25 mumol/L); losartan (10 mumol/L) eliminated SUB-induced responses. Previous studies have suggested that Ang I-convertase is important for production of Ang II in the heart. Our results are consistent with a potential role for Ang I-convertase in the production of Ang II in the vasculature, resulting in Ang II-mediated vasoconstriction.

3'-Azido-3'-deoxythymidine inhibition of human lymphocyte cytolytic function in vitro.

Despite administration of 3'-azido-3'-deoxythymidine (AZT, Zidovudine) to seriously immunocompromised patients, little has been reported regarding effects of AZT on specific immune functions. This study analyzed the in vitro effect of AZT on normal human lymphocyte cytolytic activity. AZT at concentrations up to 100 microM had no effect when added directly to cytotoxicity assays with lymphocyte effector cells and natural killer (NK)-sensitive or NK-resistant target cells. In contrast, addition of AZT to lymphocytes cultured for 4-10 days with interleukin-2 (IL-2) prior to cytotoxicity assays produced a concentration- and time-dependent inhibition; this effect was not mimicked by acyclovir or ganciclovir. Lymphocyte cell numbers and viability were not reduced in parallel to inhibition of cytolytic activity by AZT. Furthermore, AZT inhibition of IL-2-dependent cytolytic activity was not correlated with alterations in lymphocyte cell surface phenotypes by flow cytometry, and lymphocyte culture supernatant levels of interferon-gamma were not reduced by AZT. These results suggest that AZT may selectively inhibit human lymphocyte functions and thus may have implications for long-term therapeutic administration of AZT in chronic immunodeficiency states.

Impact of delayed reperfusion of myocardial hibernation on myocardial ultrastructure and function and their recoveries after reperfusion in a pig model of myocardial hibernation.

UNLABELLED: This study examined the effect of delayed reperfusion of myocardial hibernation from 24 hours to 7 days on myocardial ultrastructural and functional changes and their recoveries after reperfusion. BACKGROUND: We have previously shown in pigs that after reperfusion the functional and structural alterations in short-term myocardial hibernation which was reperfused in 24 hours can recover in 7 days. The effect of delayed reperfusion of hibernating myocardium on the extent and severity of cellular and extracellular structural changes of hibernating myocardium, and their recoveries after reperfusion is not known. METHODS AND RESULTS: A severe LAD stenosis was created in 27 pigs, reducing resting flow by 30-40% immediately after placement of the stenosis and producing acute ischemia as evidenced by regional lactate production, a decrease in regional coronary venous pH, reduced regional wall thickening (from 38.5 +/- 5.1% to 10.4 +/- 8.0%) and a 33% reduction of regional oxygen consumption. The stenosis was maintained either for 24 hours in 9 pigs (group 1) with LAD flow of 0.65 +/- 0.13 ml/min/g (38% reduction), or for 7 days in 17 pigs (group 2) with LAD flow of 0.67 +/- 0.14 ml/min/g (36% reduction). There were no differences (p = NS) in the reduction of wall thickening, rate-pressure product, lactate production, or regional oxygen consumption between group 1 and group 2. Quantitative morphometric evaluation of the ultrastructure on electromicrographs revealed a greater decrease in sarcomere volume and a higher incidence of myocytes with reduced sarcomere volume in 7-day than in 24-hour hibernating regions (53 +/- 19% versus 33 +/- 14%, p < 0.05). Patchy myocardial necrosis with replacement fibrosis was common, but 6 of the 18 pigs had no myocardial necrosis or replacement fibrosis in the 7-day hibernating group, and 4 of 9 pigs had no patchy myocyte necrosis in the 24 hour hibernating group. In 6 pigs in group 1 in which the stenosis was then released and hibernating myocardium reperfused in 24 hours, regional wall thickening recovered to 30 +/- 6% (p = NS compared to baseline) after one week of reperfusion. In 12 pigs in group 2 in which the stenosis was released and hibernating myocardium reperfused in 7 days, regional wall thickening recovered slowly, from 10.1 +/- 7.2% to 18.1 +/- 8.3% at one week (n = 5) and to 28.0 +/- 3.6% at 3-4 weeks of reperfusion (n = 7, p < 0.05 compared to baseline). Similarly, the sarcomere volume or myofilament recovered significantly (p < 0.01) and was not different compared to the normal region (p = NS) in the 24-hour hibernating region of group 1, but the recovery was much slower and was incomplete at 4 weeks (p < 0.01) compared to baseline in the 7-day hibernating region of group 2. Recovery of regional wall thickening correlated with ultrstructural recovery (p < 0.01). By multivariate stepwise regression analysis, the degree of LAD flow reduction, the extent of fibrosis, and myofilament loss were independent predictors of the extent of functional recovery. CONCLUSIONS: In a porcine model of myocardial hibernation with myocardial hypoperfusion, systolic dysfunction, and metabolic adaptations, a longer period of myocardial hibernation with delayed reperfusion was associated with more severe abnormalities of myocytes. an increasing interstitial fibrosis, and more protracted myofibrillar and functional recoveries after reperfusion. The extent of functional recovery is related to the degree of coronary flow reduction, the severity of the ultrastructural changes, and the extent of interstitial fibrosis.

Distortion of calculated whole-body hematocrit during lower-body immersion in water.

We found a difference between the venous hematocrits of immersed and nonimmersed arms during immersion of the lower body in cold water but not during a comparable exposure to warm water. Fourteen healthy men were exposed to three different experimental conditions: arm immersion, body immersion, and control. The men always sat upright while both upper extremities hung vertically at their sides. During arm immersion, one forearm was completely immersed for 30 min in either cold water (28 degrees C, n = 7) or warm water (38 degrees C, n = 7). This cold-warm water protocol was repeated on separate days for exposure to the remaining conditions of body immersion (immersion of 1 forearm and all tissues below the xiphoid process) and control (no immersion). Blood samples were simultaneously drawn from cannulated veins in both antecubital fossae. Hematocrit difference (Hct diff) was measured by subtracting the nonimmersed forearm's hematocrit (Hct dry) from the immersed forearm's hematocrit (Hct wet). Hct diff was approximately zero when the men were exposed to the control condition and body immersion in warm water. In the remaining conditions, Hct wet dropped below Hct dry (P less than 0.01, 3-way analysis of variance). The decrements of Hct diff showed there were differences between venous hematocrits in immersed and nonimmersed regions of the body, indicating that changes of the whole-body hematocrit cannot be calculated from a large-vessel hematocrit soon after immersing the lower body in cold water.

Relationship among cardiac output, shunt, and inspired O2 concentration.

In comparing gas exchange responses of the methacholine- (MCh) challenged mongrel dog with leukotriene receptor blockers and placebo at different inspiratory O2 fractions (FIO2), we previously noted systematically different values of cardiac output as a function of drug administration and/or FIO2. This confounds identification of the effects of FIO2 and/or drugs on gas exchange, because shunt is well known to vary directly with cardiac output when other factors are equal. Accordingly, in six dogs we examined the dependence of combined shunt and low ventilation-perfusion (VA/Q) blood flow ("shunt") on cardiac output in the MCh-challenged mongrel dog. Two dogs breathed 100% O2, another two breathed room air, and the final pair breathed 12% O2 while cardiac output was altered several times by sequentially opening and closing arteriovenous fistulas every 10 min for approximately 90 min after a standard MCh challenge. On 100% O2, shunt increased by 11.0% of the cardiac output per 1-l/min increase in cardiac output. On room air, the value was 7.4%. With 12% O2 breathing shunt rose by only 2.2% per 1-l/min rise in blood flow. This FIO2 -dependent behavior of the shunt-cardiac output relationship was highly reproducible, both within and between animals. It suggests that the increase in shunt with cardiac output depends more on vascular tone of noninjured areas than on tone of the low VA/Q regions (which are hypoxic at all FIO2 values).

Pulmonary and leg VO2 during submaximal exercise: implications for muscular efficiency.

Insights into muscle energetics during exercise (e.g., muscular efficiency) are often inferred from measurements of pulmonary gas exchange. This procedure presupposes that changes of pulmonary O2 (VO2) associated with increases of external work reflect accurately the increased muscle VO2. The present investigation addressed this issue directly by making simultaneous determinations of pulmonary and leg VO2 over a range of work rates calculated to elicit 20-90% of maximum VO2 on the basis of prior incremental (25 or 30 W/min) cycle ergometry. VO2 for both legs was calculated as the product of twice one-leg blood flow (constant-infusion thermodilution) and arteriovenous O2 content difference across the leg. Measurements were made 3-5 min after each work rate imposition to avoid incorporation of the VO2 slow component above the lactate threshold. For all 17 subjects, the slope of pulmonary VO2 (9.9 +/- 0.2 ml O2.W-1.min-1) was not different (P greater than 0.05) from that for leg VO2 (9.2 +/- 0.6 ml O2.W-1.min-1). Estimation of "delta" efficiency (i.e., delta work accomplished divided by delta energy expended, calculated from slope of VO2 vs. work rate and a caloric equivalent for O2 of 4.985 cal/ml) using pulmonary VO2 measurements (29.1 +/- 0.6%) was likewise not significantly different (P greater than 0.05) from that made using leg VO2 measurements (33.7 +/- 2.4%). These data suggest that the net VO2 cost of metabolic "support" processes outside the exercising legs changes little over a relatively broad range of exercise intensities. Thus, under the conditions of this investigation, changes of VO2 measured from expired gas reflected closely those occurring within the exercising legs.

Effect of inspired O2 concentration on leg lactate release during incremental exercise.

The normal rate of blood lactate accumulation during exercise is increased by hypoxia and decreased by hyperoxia. It is not known whether these changes are primarily determined by the lactate release in locomotory muscles or other tissues. Eleven men performed cycle exercise at 20, 35, 50, 92, and 100% of maximal power output while breathing 12, 21, and 100% O2. Leg lactate release was calculated at each stage of exercise as the product of femoral venous blood flow (thermodilution method) and femoral arteriovenous difference in blood lactate concentrations. Regression analysis showed that leg lactate release accounted for 90% of the variability in mean arterial lactate concentration at 20-92% maximal power output. This relationship was described by a regression line with a slope of 0.28 +/- 0.02 min/l and a y-intercept of 1.06 +/- 0.38 mmol/l (r2 = 0.90). There was no effect of inspired O2 concentration on this relationship (P > 0.05). We conclude that during continuous incremental exercise to fatigue the effect of inspired O2 concentration on blood lactate accumulation is principally determined by the rate of net lactate release in blood vessels of the locomotory muscles.

Effects of hyperoxia on maximal leg O2 supply and utilization in men.

We studied O2 transport in the leg to determine if hyperoxia will increase the maximal rate of O2 uptake (VO2max) in exercising muscle. An increase in inspired O2 fraction (FIO2) from 0.21 to 1.00 was postulated to have the following effects: 1) increase the leg VO2max by approximately 5-10%, 2) increase the maximal O2 delivery [arterial O2 concentration.flow (CaO2.Q] by approximately 10%, and 3) raise the leg VO2max in proportion to both the femoral venous PO2 and mean leg capillary PO2. To test these hypotheses, 11 men performed cycle exercise to the highest work rates (WRmax) they could achieve while breathing 100% O2 (hyperoxia), 21% O2 (normoxia), and 12% O2 (hypoxia). Leg VO2 was derived from duplicate measurements of femoral venous blood flow and CaO2 and femoral venous blood O2 concentrations (CVO2) at 20, 35, 50, 92, and 100% WRmax in each FIO2. Femoral venous leg Q (Qleg) was measured by the constant-infusion thermodilution technique, and leg O2 uptake (VO2) was determined by the Fick principle [VO2 = Qleg(CaO2-CVO2)]. Leg VO2max was the mean of duplicate values of VO2 at 100% WRmax for each FIO2. Hyperoxia increased leg VO2max by 8.1% (P = 0.016) and maximal O2 delivery by 10.9% (P = 0.05) without changing Qleg. There was a significant increase in femoral venous PO2 (P < 0.001) that was proportionally greater than the increase in leg VO2max. The results support our first and second hypotheses, providing direct evidence that in normal subjects leg VO2max is limited by O2 supply during normoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between body and leg VO2 during maximal cycle ergometry.

It is not known whether the asymptotic behavior of whole body O2 consumption (VO2) at maximal work rates (WR) is explained by similar behavior of VO2 in the exercising legs. To resolve this question, simultaneous measurements of body and leg VO2 were made at submaximal and maximal levels of effort breathing normoxic and hypoxic gases in seven trained male cyclists (maximal VO2, 64.7 +/- 2.7 ml O2.min-1.kg-1), each of whom demonstrated a reproducible VO2-WR asymptote during fatiguing incremental cycle ergometry. Left leg blood flow was measured by constant-infusion thermodilution, and total leg VO2 was calculated as the product of twice leg flow and radial arterial-femoral venous O2 concentration difference. The VO2-WR relationships determined at submaximal WR's were extrapolated to maximal WR as a basis for assessing the body and leg VO2 responses. The differences between measured and extrapolated maximal VO2 were 235 +/- 45 (body) and 203 +/- 70 (leg) ml O2/min (not significantly different). Plateauing of leg VO2 was associated with, and explained by, plateauing of both leg blood flow and O2 extraction and hence of leg VO2. We conclude that the asymptotic behavior of whole body VO2 at maximal WRs is a direct reflection of the VO2 profile at the exercising legs.