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Impaired binding of anionic drugs to serum albumin in patients with uremia is thought to be due to the accumulation of endogenous substances that bind to albumin. In this study the displacement by the anionic drugs diazepam, warfarin, and salicylic acid, which are known to be representative drugs for the binding sites on the albumin molecule, of several endogenous ligands that bind to albumin in uremic serum was examined. The free fractions of the ligands bound to albumin were separated by ultrafiltration in the presence and the absence of test drugs and assayed by high-performance liquid chromatography. Diazepam displaced indoxyl sulfate (IS), hippuric acid (HA), and indole-3-acetic acid (IAA), and warfarin displaced IS, HA, ISAA, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid from serum albumin. However, salicylic acid did not displace the substance examined. The methods reported here are useful for determining the binding sites of the endogenous ligands on albumin and to clarify the drug-ligand interaction on albumin molecule in uremic serum.

Functional omicron-iodohippurate scintigrams were obtained in 18 hypertensive patients. Each patient was examined in the prone position and during exercise. An exercise-induced transient, bilateral, hippurate transport disturbance was sought as an expression of an exercise-mediated cortical perfusion abnormality. The study sought to test the hypothesis that patients who present evidence for an exercise-induced renal perfusion disturbance would have stabilized hypertension that was no longer surgically curable because of morphological changes of the peripheral vasculature. All 18 patients continued on to therapy: 13 proceeded to renovascular reconstructive surgery, 2 had a unilateral nephrectomy, and 3 were treated with percutaneous transluminal renal angioplasty. During preoperative exercise renography, evidence of bilateral renal dysfunction developed in 10 of 18 hypertensive patients during ergometric stress (abnormal exercise response). Following surgical therapy nine of these patients with abnormal exercise scintigrams continued to have hypertensive disease, while one patient was cured. The exercise renograms of eight hypertensive patients were not influenced by the exercise protocol, and operation cured seven of these eight patients. The results suggest that an accentuated vascular response to exercise occurs in the maintenance phase of renovascular hypertension, a disturbance not observed while the hypertension is curable by surgical therapy.

The ability to convert cyclohexanecarboxylic acid to hippuric acid has been studied in liver from guinea pigs, rabbits, rats and mice using a gas chromatographic - mass spectrometric method employing selected ion monitoring. Guinea pig liver showed the highest activity, giving values double of those found in rabbit liver and five times those in rat liver. Only very weak activity was found in mouse liver. (Hydroxymethyl)cyclohexane, cyclohexanealdehyde and alpha-hydroxyethylcyclohexane, which are structurally related to cyclohexanecarboxylic acid but lack the carboxyl group, were not aromatized by guinea pig liver mitochondria. This finding indicates that the carboxyl group is essential for aromatization. Absence of aromatization was also found with the homologs cyclohexaneacetic acid and cyclohexanepropionic acid and with the di-acids trans-1,2- and trans-1,4-cyclohexanedicarboxylic acid. The effect of a methyl group in cyclohexanecarboxylic acid depended on its position. 2-Methyl-1-cyclohexanecarboxylic acid was not aromatized, however the 3- and 4-methyl derivatives underwent aromatization and subsequent conjugation with glycine. The rates of formation of m-methyl- and p-methylhippuric acid were 16% and 9%, respectively, of that found for hippuric acid from cyclohexanecarboxylic acid (8.0 nmol/min/mg protein).

Renal metabolism of salicylic acid (SA) to salicyluric acid (SU), as well as the metabolism of SU to SA, was demonstrated in the isolated perfused rat kidney. SU formation was dependent upon the inclusion of a glycine pool and glycine concentration influenced the rate of SU excretion. The total conversion of SA to SU in 1 hr was 7.7% after the administration of 1 mg SA. Administration of increasing amounts of SA diminished this extent of SU formation. The addition of a competitive substrate, benzoic acid, produced a rapid formation and excretion of hippuric acid with a corresponding inhibition of SU formation and excretion. It is important to note that when isolated kidneys were perfused with 2.5 mg SU, 20--30% of the dose was metabolized to SA. Increasing the dose of SU to 10 mg decreased the amount of SA formed. In view of observed reversible SA/SU metabolism, a larger renal contribution to overall salicylate disposition is suggested.

The active transport of organic acids in proximal renal tubules of the Campbell, Wistar and random-bred rats was studied by contact microfluorometry with anion dye fluorescein as a marker, on 1, 2, 3, 5, 10, 30 and 90 days after a unilateral nephrectomy. Sham-operated rats were used as a control. Transport was investigated under certain conditions when fluorescein was transported by Na-dependent (at 30 degrees C) or Na-independent (at 20 degrees C) systems. It is shown that fluorescein accumulation in renal tubules of operated random-bred rats at 30 degrees C is (if compared to the corresponding control) 81, 105 and 75% on 1, 2 and 3-10 days, resp., and about 90% on 30 and 90 days. Fluorescein accumulation at 20 degrees C in all investigated rats was about 90% of the corresponding control. A specific poison of Na+, K+-ATPase = ouabain inhibited fluorescein accumulation on day 1 after sham-operation by 70% to control, but there was no inhibition on day 1 after the operation (unilateral nephrectomy). P-aminohippuric acid in both media inhibited fluorescein accumulation in renal tubules of sham-operated rats better than in operated rats. It is concluded that either the amount of the carried of transport system for organic acids decreased in membranes of renal tubules of operated rats, or the affinity of carrier to organic acids is aggravated.

A rapid test to detect the hydrolysis of sodium hippurate by beta-hemolytic streptococci within 2 h was developed. All group B streptococci tested were positive using this method and all other groups were negative.

Average steady-state serum salicylate concentrations and salicyluric acid (SU) formation rates were measured in 4 subjects with rheumatoid arthritis. After a salicylate washout period (1 month), the mean observed maximum formation rate of SU was determined by collecting frequent urine samples after a single oral dose of salicylate (35 mg/kg). The patients were then commenced on appropriate high dose salicylate therapy. Two and 5 weeks later, the mean observed maximum rates of SU formation were re-determined along with the average steady-state serum salicylate concentrations. Mean observed maximum SU excretion rates increased significantly between the single-dose study (0.96 +/- 0.22 mg/kg/h) and the last dose of the high dose therapy at 2 weeks (1.65 +/- 0.30 mg/kg/h; p less than 0.01); however, there was no further increase at week 5. Similar increases in the theoretical maximum rate of SU formation (Vmax) were observed between the single-dose study and after 2 weeks of high dose salicylate therapy. Average steady-state serum salicylate concentrations showed no decline between weeks 2 and 5. High dose salicylate therapy leads to acceleration of the rate of SU formation in patients with rheumatoid arthritis and this occurs largely during the first 2 weeks of therapy.

Iodipamide is known to be actively taken up in vitro by the rabbit iris-ciliary process preparation. This uptake is partly resistant to high concentrations of hippurate and the resistant part has been called the 'liver-like' system. In vivo iodipamide is eliminated from the rabbit eye after injection into the vitreous by a saturable process. This process is hippurate-sensitive and no role for any hippurate-resistant system was found. Two explanations for the discrepancy between the results in vitro and in vivo are offered: (1) Iodipamide may be a less than perfect model substance for physiological compounds that normally are transported by a liver-like system from the vitreous cavity and the posterior aqueous humour to the blood. (2) Iodipamide is a model for compounds that are taken up by the non-pigmented epithelium of the ciliary processes by a liver-like system and transported to the pigmented epithelium for metabolic modification.

Isolated hepatocyte suspensions from rat, rabbit, dog, squirrel monkey and human livers were used to study the metabolism of amphetamine (AMP), a drug for which species-dependent differences in metabolism have been demonstrated in vivo. Hepatocytes were isolated by perfusion of the whole liver or of biopsy specimens. In general, the metabolite profile of hepatocytes from each species corresponded to the profile of urinary metabolites identified previously. Rat hepatocytes primarily metabolized AMP by aromatic hydroxylation to p-hydroxyamphetamine. Rabbit hepatocytes converted AMP almost exclusively to products of the oxidative deamination pathway. Metabolism by hepatocytes from the other three species was mixed, but oxidative deamination was somewhat more active than aromatic hydroxylation in dog, squirrel monkey and human hepatocytes. The overall rate of AMP metabolism differed significantly among the species; the half-life in the hepatocyte suspensions varied about 70-fold, with rabbit less than rat less than dog less than squirrel monkey = human. Metabolism of AMP by human hepatocytes mare closely resembled metabolism by squirrel monkey liver cells than the other species in terms of metabolite profile and rate. However, the disposition of phenylacetone, a product of oxidative deamination of AMP, varied in hepatocytes from the two primate species. Thus, the metabolism of AMP by isolated hepatocytes was unique for each species examined. These studies demonstrate the applicability of isolated hepatocytes to the study of interspecies differences in hepatic xenobiotic metabolism, providing an in vitro technique that can be readily adapted to human liver tissue.

The biotransformation of amphetaminil to amphetamine was confirmed using 3H- and 14C-labelled ampehtaminil. The metabolites were isolated, identified and quantified from blood, brain, adipose tissue and urine. These studies showed that the intact molecule of amphetaminil passes into the circulation only to a very small extent. The time spent by the amphetaminil in the alimentary canal does not appear to be a critical factor in the stability and degradation of this substance. The proportion of unchanged amphetaminil represents no more than 2% of the total radioactivity in the blood. The amphetamine, which results from the cleavage of amphetaminil, enters the CNS and is excreted in the urine after hydroxylation and glucuronidation. The other cleavage product, benzaldehyde, seems to be rapidly converted into hippuric acid, which is excreted. Amphetaminil is enriched in adipose tissue, especially after i.p. injection; but this fraction will be cleaved upon re-entering the blood, and it can only enter the brain as amphetamine.

Twenty-four strains of Mobiluncus mulieris and 27 strains of Mobiluncus curtisii were tested with respect to 6 different biochemical characteristics: arginin-decarboxylase activity, beta-galactosidase activity, synergistic hemolysis with Staphylococcus aureus, hydrolysis of hippurate, migration through soft agar and reduction of nitrate. Antigens of the same strains, prepared by ultrasonication, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting using polyclonal rabbit antisera against two of the M. mulieris strains and five of the M. curtisii strains. Two different strongly reacting protein antigens could be detected in the M. mulieris strains. These strains could be separated into three groups based on the possession of either of the two antigens or both. In the M. curtisii strains, 10 strongly reacting protein antigens could be detected. Four strains possessed only one of these antigens, one did not possess any, while the remaining strains possessed different sets of 9 of them. Within each species common protein antigens were detected. No antigens were found which were shared by both species. The biochemical characteristics studied could not differentiate between the antigenic groups in any of the species. None of the antigenic subgroups of M. curtisii found in the present study was identical with any of the two subspecies (curtisii and holmesii) which have been proposed.

The fate of salicyluric acid (SU) after oral administration was examined in rabbits. Salicylic acid (SA) and unchanged SU were detected in the blood after oral administration of SU. However, SU only was found after intravenous administration of SU, suggesting that presystemic deconjugation of glycine was involved. After treatment of rabbits with kanamycin sulfate, complete inhibition of the formation of SA after oral administration of SU was demonstrated, indicating that the intestinal microflora was responsible for the biotransformation. Furthermore, in vitro incubation of SU with contents of gut showed that the major source of the hydrolysis was the hind gut. Based on the findings described above, the time course data for blood concentration of SU and SA after oral administration of SU have been subjected to curve-fitting by a nonlinear least squares program. The SU and SA values obtained with the simplified model containing only first-order kinetic process which is proposed by the present authors were found to agree with those obtained from experiments.

The urinary metabolites of a single dose (1 g) of salicylic acid were investigated in black Nigerian subjects (78 females 44 males). Qualitatively, the major metabolites were the glycine and glucuronic acid conjugates. Quantitatively, there was a statistically significant difference in the level of these metabolites between female and male subjects (P less than 0.001) (using Student's t-test). The results of the present study compared with earlier published data show a statistically significant quantitative difference between black Nigerians and Caucasians (P less than 0.001). The results suggest possible racial and sex differences in the metabolism of salicylic acid.

Changes in 131I hippuran clearance were estimated by a non-catheter method, as well as total clearance and clearance of each kidney, in 43 women at various stages of pregnancy and in 13 non-pregnant women who were not using hormonal contraceptives. The results are demonstrated in graph form. The methods used are described in detail. The procedure for determining clearance in separate kidneys is indicated. It was found that clearance values increased up to the 35th and 36th weeks of pregnancy. The difference between the sides that were found corresponds with the previously known difference in the function of the right and left kidneys and ureters.

The use of in vitro preparations of human skin to study percutaneous absorption is widespread. Yet, up to the present time, little has been done to systematically validate this model and demonstrate the extent to which it mimicks in vivo absorption. In this study, the permeability of 12 organic compounds has been evaluated in excised skin and the results compared to those obtained previously by others in living man. With special emphasis being given here to duplicating in vivo conditions, it was possible to demonstrate an excellent qualitative agreement between the two methods. In all cases, the absorption pattern determined in vitro rather precisely paralleled the pattern which was obtained in vivo. Quantitative agreement between the two sets of data was less than perfect, although the in vitro method adequately distinguished compounds of low permeability from those of high permeability and ranked then in approximately the same order found in vivo. This systematic comparison of in vitro with in vivo data was clearly shown how accurately in vitro absorption studies can reflect the living state.

To detect whether the nephrotoxicity of cis-diamminedichloroplatinum (DDP) is acute and can be demonstrated at an early stage in man, a method for estimating the function of the kidneys during intensive hydration was devised. The method includes a calculation of the clearance of 125I-orthoiodohippurate and an estimation of the glomerular filtration rate (GFR) from fast changes of the extracellular volume (ECV) and the mean transit time of 99mTc-DTPA in this volume. We examined nine patients with testicular cancer on 2 consecutive days for acute nephrotoxicity while they were undergoing treatment with cis-platinum. Placebo was given on day 1, cis-platinum on day 2. On both days the patients were hydrated with 4 l saline, glucose, and mannitol (0.51) over a period of 4 h, which resulted in an increase of 125I-orthoiodohippurate clearance on both days (P less than 0.01). The increase was, however, lower on the day of treatment with cis-platinum than on the day with placebo (P less than 0.05). There were no acute changes in the GFR. This indicates that treatment with DDP inhibits the active transport of 125I-orthoiodohippurate in the tubules; that is to say there is an acute effect on the kidney function. There were no acute changes in the GFR, but in the long-term followup study we found that the GFR had decreased significantly (P less than 0.05) after 2 months of treatment. During the first year after the initiation of treatment the GFR changes were found to progress. A significant increase in se-creatinine was not observed until 6 months after the initiation of treatment (P less than 0.05). The degree of chronic nephrotoxicity did not correlate in individual patients with the acute changes in kidney function.

The comparative performance of three presumptive identification tests for group B haemolytic streptococci was investigated, using 371 different clinical isolates of group B streptococci. Hippurate was hydrolysed by 96-1%, the CAMP reaction was positive in 95-0%, and pigment was produced by 97-3%. A combination of any two tests would have detected over 99-8%. On bile esculin agar 99-0% were able to grow, but non hydrolysed esculin; 5-1% were susceptible to bacitracin. The minimum inhibitory concentrations of five antibiotics for 279 group B streptococci were determined. All were susceptible to penicillin G, ampicillin, cephalothin, and erythromycin, while 80% were resistant to tetracycline. The MIC distributions were independent of the results of any identification test.

Attack by .OH radicals, generated by a Fenton system, upon salicylate produces 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate as major products and catechol as a minor product. H.p.l.c. separation combined with electrochemical detection was used to identify and quantify 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate in human plasma and synovial fluid. We propose that conversion of salicylate into 2,3-dihydroxybenzoate, or of other aromatic compounds into specific hydroxylated products, may be a useful assay for .OH formation in the human body.

Recent studies have demonstrated a cytoprotective effect of sucralfate on gastric mucosa in patients receiving aspirin. The potential drug interaction between sucralfate and aspirin was evaluated in a randomized crossover manner in 12 healthy men. Subjects were initially given a single dose of aspirin alone or in combination with sucralfate for 2 days. The drug dosing schedule was then reversed after 1 week. Sixteen blood samples were drawn after each aspirin dose for HPLC assay of aspirin and its metabolites. Pharmacokinetic parameters were calculated for aspirin, salicylic acid, and salicyluric acid. None of these parameters demonstrated any statistically significant differences between the two treatment groups. The combined use of sucralfate and aspirin is therefore not likely to result in a clinically significant pharmacokinetic drug interaction. The systemic therapeutic effect of aspirin is not expected to be altered when sucralfate is used concurrently in patients receiving chronic aspirin therapy.

Hippurate function scintiscans were obtained in prone and standing positions in a group of 76 patients with concurrent hypertension and nephroptosis. TWelve of these patients had massive, bilateral disturbance of intrarenal hippurate transport in the standing position; hippurate transport was normal in the prone position. This pattern was present in only three of 120 normotensive patients with nephroptosis. To investigate the importance of nephroptosis, 87 other hypertensive patients were examined. Eighteen of these patients demonstrated posture-dependent tubular dysfunction, but only four had nephroptosis. The results suggest a direct relationship between bilateral posture-dependent tubular dysfunction and hypertension.

Two hundred eighty-five isolates of Campylobacter jejuni-Campylobacter coli from children with gastroenteritis at The Hospital for Sick Children (Toronto, Canada) over a three-year period were biotyped by the hippurate hydrolysis test and serotyped on the basis of thermostable, soluble antigens by the passive hemagglutination technique. Hippurate-negative strains (C. coli) were only 3.2% of the isolates. Ninety-seven percent of the isolates were serotypable with 55 antisera. About half of the strains belonged to one of four serotypes (2, 4, 3, or 1); about three-quarters belonged to one of 10 serotypes. Serotype 2 was consistently the commonest serotype in each of the three years of the study, accounting for 15%-20% of all isolates tested.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.

Current methods for the identification of the Legionellaceae to species level are suitable only for the specialist or research laboratory. As part of a continuing taxonomic study 42 simple biochemical tests were screened for their ability to differentiate species of Legionella. Only 23 of these were of practical use. These tests are able to differentiate 21 of 23 recognized species of Legionella and six new species. Phenotypic screening with these tests may prove useful to the routine microbiologist and be a viable alternative to identification techniques currently employed.

A gas-liquid chromatographic method which requires no chloroform extraction of the split products has been investigated for the detection of hippurate hydrolysis by Campylobacter spp. This technique gave better reproducibility than other tests also used in this study and allows the routine use of the gas-liquid chromatographic method for identification of Campylobacter isolates.

A total of 163 strains, including 106 strains of Legionella pneumophila, 28 strains of Tatlockia micdadei, and 29 strains of other legionellae (including members of the proposed genus Fluoribacter), were studied. Ten tests which together could distinguish the genera previously proposed were identified. These tests included catalase-peroxidase, gelatinase, hippurate hydrolysis, starch hydrolysis, medium browning, acetoin production, oxidase, medium fluorescence, colony fluorescence, and the bromcresol purple spot test. T. micdadei strains were strongly catalase positive and bromcresol purple spot test positive and produced acetoin but otherwise were usually inert in the other tests. L. pneumophila and Fluoribacter species could usually be distinguished by strength of catalase activity, blue-white colony fluorescence (if present), and differences in frequency of hippurate hydrolysis, starch hydrolysis, yellow-green medium fluorescence, and, to a lesser extent, oxidase activity. With a simple algorithm and computer program, the overall accuracy was 98.8%.

The blood concentrations of salicyluric acid and salicylic acid following intracecal and rectal administration of salicyluric acid were determined in rabbits. Immediate and very extensive salicylic acid formation in the cecum was found following intracecal administration. After rectal administration, a small amount of salicyluric acid was absorbed in intact form. The rest was rapidly hydrolyzed to salicylic acid, which was subsequently absorbed. The blood concentration of salicylic acid was maintained at 1.3-1.8 micrograms/ml from 2 to 12 h. Three doses of salicyluric acid were administered rectally. The peak level of salicyluric acid increased with dose. However, salicylic acid concentration in the blood following administration of salicyluric acid at 10.0 mg/kg (salicylic acid equivalent) was not double that observed following administration of salicyluric acid at 5.0 mg/kg (salicylic acid equivalent). It appears that a larger amount of salicyluric acid in the rectal lumen may have saturated the glycine deconjugation system.

The metabolism of acetaminophen and phenacetin by rabbit kidney slices was investigated. Phenacetin, like aspirin, inhibited [131I] Hippuran accumulation by the organic acid transport system. Phenacetin exhibited a dose-dependent inhibition with a K1 of 0.5 mM; acetaminophen at concentrations as high as 1 mM did not alter organic acid transport. [3H] acetaminophen slice: media ratios of approximately 1 or less suggested that acetaminophen entered cortex and outer and inner medullary slices by diffusion rather than active transport. Approximately 95% of the acetaminophen within the slices readily diffused out into the media. The chromatographic patterns of this material were similar to that of the incubation media. The acetaminophen remaining within the slices was acid-precipitable and not extractable with organic solvents. This covalent binding of acetaminophen was inhibited by glutathione. More acetaminophen was bound in the renal medullar than cortex. These data suggest that the renal metabolism of acetaminophen observed in this in vitro study may be related to the nephritis observed in analgesic abuse.

1. The action of the penicillin acylase enzyme of Escherichia coli N.C.I.B. 8743 on non-penicillin substrates suggests that the enzyme is an amidohydrolase. 2. The rates of hydrolysis for a small group of penicillins closely parallel those for a corresponding series of N-acylglycines. 3. For a series of E. coli strains, ability to cause rapid hydrolysis of phenylacetylglycine is correlated with ability to hydrolyse benzylpenicillin. 4. Amides and N-acylglycines are hydrolysed to the corresponding acids. The phenylacetyl group is hydrolysed most readily. Benzamide and beta-phenylpropionamide are not substrates. In a series of aliphatic acylglycines only valeryl- and hexanoyl-glycine are substrates. 5. Acylated l- but not d-alpha-amino acids are hydrolysed. d-alpha-Hydroxyphenylacetamide is a better substrate than the l compound.

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.

A battery of morphological, physiological, and biochemical tests, including paper chromatographic analysis of whole cell hydrolysates, was used to study 177 cultures of aerobic actinomycetes. One hundred thirty-five of the 177 were submitted as diagnostic laboratory specimens; of these, 129 were identified as belonging to 1 of 10 genus or species groups. The tests and procedures used were found to be relatively easy to perform and interpret. On the basis of the results, a flow chart was devised to permit the step by step identification of unknown clinical isolates of these organisms.