Spectrophotometry of hemoglobin: a comparison of dog and man

1. The absorptivity at 540 nm of hemiglobincyanide (epsilon 540HiCN) from dog blood was determined on the basis of iron and found to be within the range formerly obtained for human hemoglobin. 2. Consequently, epsilon 540HiCN = 11.0, the established value for human hemoglobin, may be used for dog hemoglobin. 3. On this basis the absorption spectra of oxyhemoglobin, de-oxygenated hemoglobin, carboxyhemoglobin, hemiglobin (methemoglobin) and hemiglobincyanide were determined for dog hemoglobin. 4. No significant differences were found between dog and human hemoglobin, except that dog hemiglobin binds less OH- as reflected in a difference between the absorption spectra of dog and human hemiglobin at the same pH.     

Glucagon administration induces lowering of serum T3 and rise in reverse T3 in euthyroid healthy subjects

Euthyroid sick syndrome is characterized by low serum T3 and raised reverse T3 (rT3). Most of the states with this syndrome are also documented to manifest hyperglucagonemia. Furthermore, several recent studies have suggested that glucagon may play a role in T4 monodeiodination in some of these states such as starvation and uncontrolled diabetes mellitus. Therefore, hyperglucagonemia was induced by intravenous glucagon administration in euthyroid healthy volunteers and thyroid hormone levels were determined at frequent intervals up to six hours. Plasma glucose and insulin rose promptly on glucagon administration, thus establishing the physiologic effect of glucagon. Serum T4, free T4, T3 resin uptake, and TSH concentrations remained unaltered throughout the study period. Serum T3 declined to a significantly low level (P less than 0.05) between 60-90 minutes. Serum rT3 rose significantly (P less than 0.05) by four hours and the rise was progressive till the end of the study period. Therefore, these results suggest that hyperglucagonemia may be one of the factors responsible for lowering of T3 and a rise in rT3 in euthyroid sick syndrome.     

Spectrofluorimetric determination of dipyridamole in serum — a comparison of two methods

Two spectrofluorimetric methods for the determination of dipyridamole in plasma are described. The thin-layer chromatographic—fluoridensitometric method utilizes 1 ml of plasma which is extracted at pH 10 with diethyl ether—dichloromethane (80:20). The organic phase is evaporated to dryness, reconstituted in 250 μl dichloromethane and 5 μl are spotted on a silica gel 60 plate. The plate is developed in ethyl acetate—methanol—ammonia (85:10:5), dried, dipped in a paraffin wax solution, dried, and scanned using 380 nm as excitation wavelength, a 430 nm cut-off filter, and collecting all emitted light on the photomultiplier. Quantitation was done by the external standard method, peak heights being measured and a calibration graph constructed. For the spectrofluorimetric method 1 ml of plasma is extracted at pH 10 with 8 ml of hexane—isoamyl alcohol (95:5) and the organic phase used directly for the measurement of the fluorescence intensity (excitation 405 nm, emission 495 nm). Quantitation was done by measuring the fluorescence of standards that were treated as above and constructing a calibration graph of concentration versus fluorescence intensity. Concentrations of unknowns were found by interpolation from this graph. The two methods were found to exhibit good correlation but the spectrofluorimetric method proved to be more amenable to the analysis of a large number of samples.     

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.     

Biotransformation of urapidil: isolation and identification of metabolites in mouse, rat, dog and man

The identification of biotransformation products of the new antihypertensive drug urapidil in mouse, rat, dog and man has been performed by means of high-performance liquid chromatographic and mass spectrometric techniques. In urine, three metabolites were found in addition to the unchanged drug. The para-hydroxylated product (1) (6-(3-[4-(o-methoxy-p-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyl-uracil), the O-demethylated compound (2) (6-(3-[4-(o-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyluracil) and the uracil-N-dealkylated compound (3) (6-(3-[4-(o-methoxyphenyl)piperazinyl]-propylamino)-1-methyluracil). In urine of dog, the metabolite with the N-oxide structure (5) was also identified, but only in trace amounts (6-(3-[4-(o-methoxyphenyl)piperazinyl-N-oxide]-propylamino)-1, 3-dimethyluracil).     

Serum T4, T3 and reverse T3 during treatment with propranolol in hyperthyroidism, L-T4 treated myxedema and in normal man.

Serum concentrations of T4, T3 and reverse T3 were studied in two hyperthyroid groups (n = 13 and 11), in a group of normals (n = 9) and in a group of L-T4 substituted patients (n = 7) with severe pretreatment hypothyroidism. Serum T4 did not change except in one of the hyperthyroid groups change to in which a slight decrease was found. In all groups a significant fall in serum T3 and a significant rise in serum reverse T3 were found. An expected increase in serum TSH in the normal and in the L-T4 substituted groups could not be demonstrated.     

Separation into two major forms of beta(1-3)N-acetylglucosaminyltransferase from human serum

The major beta(1-3)N-acetylglucosaminyltransferase [beta(1-3)GlcNAc-transferase] activity in human serum was isolated by DEAE- and CM-Sepharose column chromatography. This enzyme fraction consisted of two forms of the enzyme, which were separated from each other on a DEAE-Sepharose column and designated as GNAc-TI and GNAc-TII, respectively. They have the same molecular weights (about 90,000), optimum pH values (between pH 7.5 and 8.5) and apparent Km values for N-acetyllactosamine and lactose (7.1-8.8 mM and 10.9-11.5 mM, respectively). Glycosidase and methylation analyses of the reaction products demonstrated that both the enzyme catalyze exclusively the transfer of one N-acetylglucosamine to position C-3 of the terminal galactose of lactose in the beta linkage.     

How to obtain NNT from Cohen's d: comparison of two methods

Background

In the literature we find many indices of size of treatment effect (effect size: ES). The preferred index of treatment effect in evidence-based medicine is the number needed to treat (NNT), while the most common one in the medical literature is Cohen''s d when the outcome is continuous. There is confusion about how to convert Cohen''s d into NNT.

Methods

We conducted meta-analyses of individual patient data from 10 randomized controlled trials of second generation antipsychotics for schizophrenia (n = 4278) to produce Cohen''s d and NNTs for various definitions of response, using cutoffs of 10% through 90% reduction on the symptom severity scale. These actual NNTs were compared with NNTs calculated from Cohen''s d according to two proposed methods in the literature (Kraemer, et al., Biological Psychiatry, 2006; Furukawa, Lancet, 1999).

Results

NNTs from Kraemer''s method overlapped with the actual NNTs in 56%, while those based on Furukawa''s method fell within the observed ranges of NNTs in 97% of the examined instances. For various definitions of response corresponding with 10% through 70% symptom reduction where we observed a non-small number of responders, the degree of agreement for the former method was at a chance level (ANOVA ICC of 0.12, p = 0.22) but that for the latter method was ANOVA ICC of 0.86 (95%CI: 0.55 to 0.95, p<0.01).

Conclusions

Furukawa''s method allows more accurate prediction of NNTs from Cohen''s d. Kraemer''s method gives a wrong impression that NNT is constant for a given d even when the event rate differs.     

Whole-system estimation of denitrification in a plains river: a comparison of two methods

Whole-system denitrification in the South Platte River was measured over a 13-month period using an open-channel N₂ method and mass-balance measurements. Concentrations of dissolved N₂ were measured with high precision by membrane-inlet mass spectrometry and estimates of denitrification were based on the mass flux of N₂, after correction for reaeration and groundwater flux. Open-channel estimates of denitrification ranged from 0 to 3.08 g N m^–2 d^–1 and the mean annual rate was 1.62 g N m^–2 d^–1, which corresponds to removal of approximately 34% of the nitrate transported by the river over a distance of 18.5 km. Over the same period of time, estimates of denitrification based on mass-balance measurements ranged from 0.29 to 5.25 g N m^–2 d^–1 and the mean annual rate was 2.11 g N m^–2 d^–1. The two methods revealed similar seasonal patterns of denitrification the highest rates were measured from late April to August and the lowest rates were in winter. Both methods provide whole-system estimates of denitrification in running waters; where reaeration rate coefficients are low and flux of groundwater is well quantified, the open-channel method has fewer sources of uncertainty and is easier to implement.     

Separation of arsenic metabolites in dog plasma and urine following intravenous injection of 74As

A simple chromatographic method to separate the arsenic metabolites in plasma and urine following iv administration of ⁷⁴As to dogs is described. Plasma and urine samples were chromatographed on a cation-exchange resin column. Inorganic, monomethylated and dimethylated arsenic compounds were eluted with 0.5 n HCl, H₂O, and 20% ($\text{v}\text{v}$) NH₄OH solution respectively. Experiments on two dogs slow ～90% of the ⁷⁴As in urine present in the form of dimethylarsinic acid 3 days after administration. It is also the predominant species in plasma 2 hr after dosing.     

Microbiological air analysis in dental surgeries: a comparison between two methods

The microbiological quality of indoor air is creating an increasing interest especially as far as places at risk such as hospitals, clinics, medical and odontological surgeries are concerned. Working with the odontologists of our province we have been carrying out a research aimed at preventing cross-infection in odontology. Data obtained from the microbiological analysis of the air in 36 surgeries using S.A.S. were discussed during the V National Congress of Aerobiology. During that congress the need of setting a standardized technique of air sampling in indoor environments emerged and two routes have been identified: (1) the gravimetric technique on open plate exposed for an hour close to the dental unit and (2) the use of the volumetric sampler which gives qualitative data expressed as colonies forming units per cubic metre of air. However, both of these techniques present some problems: using the first a loss of micro-organisms has been noticed due to the variability of the air fluxes and the different weight of the biological particles; using the second one the bacterial charge is also undervalued, because of the stress suffered by the bacteria with the use of the volumetric sampler. In the light of these statements we decided to use both in dental surgeries to be able to compare the results obtained. Our project is expected to carry out at least one inspection and the relative sampling (indoor air, water of the dental unit, air of the syringe, disinfectant solution, surface tampons, biological test of sterility) in each dental surgery in the territory of our health Unit, located in Ferrara, Northern Italy.     

Rate of recalls in congenital hypothyroidism based upon a regional survey in Isfahan, Iran, using serum T4 and TSH analyses: comparison of two different recall methods

AIMS: To evaluate and compare the recall rate in congenital hypothyroidism screening project in Isfahan, first using an approach involving measures of both TSH and T4 and then using TSH alone. METHODS: From June 2002 to January 2005, serum TSH and T4 level of referred neonates were measured at 3rd to 7th day of birth through venous sampling. If neonates' serum TSH was >20 mIU/l or T4 was <6.5 microg/dl by the first protocol, or TSH was >20 mIU/l by the second protocol, they were recalled. TSH and T4 were measured using an immunoradiometric assay and radioimmunoassay, respectively. Neonates with TSH > 10 and T4 < 6.5 on their second measurement were considered as congenitally hypothyroid. RESULTS: Serum T4 and TSH of 29,425 neonates by first and 57,235 neonates by second recall approach were measured. Recall rate was higher in the first protocol (2.2% vs. 0.6%, p < 0.05). Most of the recalled neonates in the first protocol were recalled for low T4 level (p < 0.05). The prevalence of CH was 1 in 350 livebirths. CONCLUSION: Although the recall rate was in the acceptable range by either approach, the TSH alone protocol seems to be a more sensitive and practical approach with the least recall burden and considering the high prevalence of CH in our region merit adaptation of widespread screening for CH using TSH measurements from heel stab blood spotted on filter paper.     

Quantitative in vivo receptor binding IV: Detection of muscarinic receptor down-regulation by equilibrium and by tracer kinetic methods

Newly-developed methods for estimation of in vivo binding to neurotransmitter receptors should enable the detection and quantification of physiologic or pathologic changes in receptor numbers. In the present study, both equilibrium and kinetic experimental strategies for in vivo muscarinic receptor determination were applied to the detection of receptor changes induced by chronic inhibition of acetylcholinesterase in the rat. Following one week of treatment, in vitro receptor autoradiography utilizing [³H]scopolamine revealed significant losses of muscarinic binding in the cerebral cortex, hippocampus, striatum and in cranial nerve motor nuclei. The in vivo distribution of [³H]scopolamine, following infusion to approach equilibrium binding in the brain, revealed reductions in binding which paralleled the pattern and magnitude of changes detected in vitro. A simplified tracer kinetic estimation following bolus injection of the ligand also detected substantial reductions in forebrain muscarinic receptor binding. These results indicate the feasibility of detecting receptor changes underlying neuropathologic conditions in vivo, and suggest that either equilibrium or kinetic experimental approaches may be extended to clinical research applications with the use of positron or single-photon emission tomography.Special issue dedicated to Dr. Louis Sokoloff.     

The analysis of metabolites in rainbow trout white muscle: a comparison of different sampling and processing methods

We have investigated the effects of different sampling and processing methods on metabolite concentrations [glycogen (Gly), glucose (Glu), lactate (Lac), pyruvate (Pyr), ammonia (Amm), creatine phosphate (PCr), creatine (Cr), and adenosine triphosphate (ATP)] measured in white muscle of rainbow trout at rest and immediately after exhaustive exercise. When samples were taken from resting fish by rapid needle biopsy (without anaesthesia), direct freezing of the needles in liquid N₂ yielded lower Lac and Glu levels than if the muscle cores were quickly blown out into liquid N₂. However, killing of the fish by an overdose of MS-222 followed by freeze-clamping of excised muscle was superior to the biopsy method in preserving high levels of PCr and Gly (91 and 62% higher, respectively). In parallel, the MS-222 method also yielded lower levels of Amm (80%) and Lac (47%). Samples freeze-clamped by the MS-222 method were used to evaluate three methods of subsequent processing for enzymatic analysis of metabolites: classic glass homogenization (GH) in 8% perchloric acid (PCA) c. mortar and pestle (MP) pulverization or freeze-drying (FD) prior to PCA extraction. For all metabolites, GH and MP methods produced similar values. However, the FD technique yielded 20% higher PCr levels which represented over 80% phosphorylation of the total Cr pool at rest, the highest ever reported via enzymatic analysis. Glu was also higher by FD, bul Gly, Lac, and ATP were not affected. Indeed ATP was relatively stable throughout all sampling and processing procedures. MP, GH, MP&GH combination, and high speed motor driven grinding techniques all yielded similar Amm levels in resting muscle. However, tests demonstrated that even brief thawing of tissue greatly elevated Amm, while FD resulted in artificially low Amm values due to evaporative losses during lyophilization. Overall, muscle sampling by freeze-clamping on trout killed by MS-222 overdose, followed by FD prior to PCA extraction, appears to be the best combination for the measurement of all white muscle metabolites except Amm, for which MP or GH are preferable.