Reducing pathology test misuse.

Our laboratory serves a 450-bed acute care hospital in the rapidly growing Gold Coast region. The problem facing the department comprised an almost 10 per cent yearly increase in patient numbers in the face of budgetary constraints and pressure to increase efficiency. In January and February 1992 a vigorous effort was made to reduce pathology test numbers without compromising quality of patient care. The approach adopted involved targeting 'unnecessary' tests, defined as those investigations that would not affect a clinician's management of the patient. A team effort by laboratory scientific staff and heads of clinical departments was aimed at scrutinizing the ordering patterns of junior medical staff, who generate most of the test requests. The result was a 'test per patient' figure of 8.2 for both months, calculated as the number of tests on each request form received. In comparison, for January and February of the preceding four years, 1988 to 1991, the number of tests per patient fluctuated between 9.9 and 11.8. Patterns of unnecessary ordering that emerged included duplicate ordering of identical tests due to poor communication between house-staff, too frequent repeats of tests (for example, daily liver function tests), crossmatch where low transfusion likelihood would make group-and-hold appropriate, tests generated by nursing staff, and numerous others where the result would not affect management (or indeed even be looked at!). The nearly 20 per cent drop in tests does not include those instances where all tests on a request form were disallowed and the encounter therefore not included as a 'patient' statistic.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine turnover in various tissues at rest and during exercise: evidence for a training effect.

This study quantitates the sympathetic response to an acute bout of exercise in specific tissues recruited during exercise in Fischer 344 rats (National Institute on Aging, Bethesda, MD), as well as determining any associated training adaptations. After 10 weeks of treadmill running, significant improvements were found for both endurance time and running speed eliciting maximal response in trained animals compared to controls (477% and 57%, respectively). Norepinephrine turnover (NEt) was determined by the administration of alpha-methyl tyrosine (250 mg/kg), a competitive inhibitor of tyrosine hydroxylase. Animals were killed at rest, after 30 minutes of submaximal (75% max) and at maximal exercise. Cardiac NEt values were 36.3, 141.5 and 528.1 ng/g/h for trained (Tr), 37.2, 134.5 and 638.7 ng/g/h for untrained (Un) animals at rest, submax and max, respectively. A similar response was found in the adrenals. However, the opposite was found with training in the liver. NEt values in Tr animals were greater (6.4, 80.5 and 161.8 ng/g/h) compared with Un (5.8, 25.5 and 75.8 ng/g/h) at rest, submax and max, respectively. It was concluded that training elicits a diminished sympathetic response in the heart and adrenals in response to an acute bout of exercise. Such a training effect was not found in the liver.

Practice patterns of family physicians with 2-year residency v. 1-year internship training: do both roads lead to Rome?

Are there differences in patterns of practice between actively practising physicians who have been certified after a 2-year family practice residency and matched physicians without certification who have completed the standard 1-year internship? With the use of billing files prepared by the British Columbia Medical Association a group of 65 family practice certificants in active practice in British Columbia was compared with a control group of 130 internship trainees matched by year and school of graduation, category of billing (i.e., solo or group) and region. A wide range of practice features was assessed for the fiscal years 1984-85, 1985-86 and 1986-87. No differences were detected between the groups in 1986-87 for the following practice variables: number of patients (1888 and 1842 respectively), number of personal services billed for (7265 and 7173), number of personal services per patient (3.9), amount of funding for personal services ($140,192 and $140,100) and amount per patient for personal services ($77 and $79). Age-adjusted costs for male and female patients were similar in the two groups. Of six services thought to be influenced by type of training, only maternity care generated a significantly higher number of billings in the study group (341 v. 249). These results suggest that there is no demonstrable effect of training on patterns of practice. However, the question of the effect of training on quality of care and whether the 2-year residency may have a longer effect on practice patterns should be the focus of future research.

Sympathetic response to exercise in various tissues with advancing age.

It was the purpose of this investigation to examine any age-related changes in norepinephrine turnover (NEt) in four tissues at rest and during exercise. Fischer 344 rats 6 (n = 20) and 25 mo of age (n = 20) were received from the National Institute on Aging. NEt was determined at rest, during 30 min of submaximal exercise, and at maximal exercise by administration of alpha-methyl-p-tyrosine, a competitive inhibitor of tyrosine hydroxylase. Resting NE declined with age in both heart (38.2 vs. 30.5 ng.g-1.h-1) and liver (11.2 vs. 6.4 ng.g-1.h-1). NEt was greater in the older animals compared with the young animals in heart (120.9 vs. 169.5 ng.g-1.h-1), liver (23.1 vs. 38.9 ng.g-1.h-1), and adrenals (74.0 vs. 98.4 ng.mg-1.h-1) during submaximal exercise. In response to maximal exercise, NEt varied depending on age and tissue. It was concluded that, in response to exercise stress, the older animals generally demonstrated a higher NEt (reflecting elevated sympathetic activity) perhaps because of a decreased adrenergic receptor sensitivity and/or responsiveness.

Guanethidine N-oxidation in human liver microsomes.

The capacity of human liver microsomes to N-oxidize guanethidine from 25 subjects has been assessed. Guanethidine N-oxidation was optimal at pH 8.5 and proceeded at only 16% of the maximal rate at pH 7.4. The mean rates of guanethidine N-oxidation at pH 8.5 and 7.4 were 2.46 +/- 0.89 (mean +/- s.d., n = 25) and 0.38 +/- 0.22 (mean +/- s.d., n = 22), respectively. Interindividual differences in the rate of guanethidine N-oxidation at pH 8.5 and 7.4 were 17- and 11-fold, respectively. The cytochrome P450 inhibitors, proadifen and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), at both pH 8.5 and 7.4 caused less than 20% reduction in the rate of guanethidine N-oxidation by human liver microsomes. These data indicate that guanethidine N-oxidation can be used as a measure of flavin-containing monooxygenase activity in human liver.

Effects of substance P and neurokinin A (substance K) on motor behavior: unique effect of substance P attributable to its amino-terminal sequence.

The effects of intraventricular injections of the neuropeptides substance P (SP) and neurokinin A (NK-A; also called substance K) on spontaneous motor behavior were examined in mice. SP and NK-A were essentially equipotent at enhancing grooming and scratching behavior, and at reducing sniffing behavior. However, SP significantly enhanced hindlimb rearing behavior, while NK-A reduced this behavior. The effects of 3 other tachykinins, physalaemin, eledoisin and kassinin, were comparable to those of NK-A, including the reduction in rearing. Thus, SP is unique among tachykinins in its potentiation of rearing behavior. It was further demonstrated that carboxy-terminal SP fragments with tachykinin activity on smooth muscle resemble NK-A, and not SP, in their effects on motor behavior. In contrast, amino-terminal SP fragments, devoid of tachykinin-like activity, reproduced the one motor effect unique to SP, enhanced rearing, while lacking those actions common to all tachykinins. The structural requirements for enhanced rearing behavior by amino-terminal fragments were quite specific, in terms of chain length and sensitivity to D-amino acid substitutions, with the natural amino-terminal hexa- and heptapeptides being most active. The implications of these findings are discussed in light of recent observations that these same amino-terminal SP fragments are produced in vivo as metabolites of SP.

Benzodiazepine abuse.

Benzodiazepine abuse is indicated by excessive and long-term consumption, particularly by the elderly. Side-effects associated with long-term use are reviewed with special reference to the problem of dependence. Factors predictive of such dependence are examined and procedures for its management are described.

Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase.

The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450.

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.

Effect of microsomal enzyme inducers on the urinary excretion pattern of mutagenic metabolites of the carcinogen 2,4-toluenediamine.

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450.

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.

Age and strain differences in some behavioral effects of intracranial substance P.

Intracerebroventricular (ICV) injections of substance P (SP) induce a vigorous reciprocal hindlimb scratching (RHS) syndrome, accompanied by extensive grooming behavior. There is a significant (approximately 1000X) difference in responsiveness to SP, as measured by RHS and grooming, in mice as a function of genetic strain (Swiss/Webster, C57 or DBA) and age. There was considerable specificity in the ability of drugs to increase responsiveness in the least responsive type of mouse (aged DBA/2J). Responding in old DBAs was enhanced by high doses of naloxone, suggesting the involvement of opioid peptides. Significant enhancement of responding by alpha-methyl tyrosine and propranolol, but not by phenoxybenzamine or haloperidol, indicated that beta-adrenergic systems are also involved. Similar manipulations of serotonergic systems were without effect.

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene.

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) have previously been shown to induce dose-dependent DNA strand breaks in primary hepatocytes from mice and rats. In an attempt to determine the relationship between the extent of DNA strand breaks and the formation of specific DNA-carcinogen bound adducts in murine liver, the capability of N-OH-AAF and N-OAc-AAF to induce both DNA single strand breaks and adduct formation in in vivo and in primary hepatocytes was measured. N-OH-AAF induced a low level of DNA damage in F344 rats (10 mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment. The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene (Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene (Gua-C8-AF) 34% versus 67% and 3-(guanin-N2-yl)-2-acetylaminofluorene (Gua-N2-AAF) 11% versus 10%, respectively, for rat and mouse liver. An additional unknown adduct (12%) was detected in mouse liver. Dose dependent DNA binding and formation of individual DNA adducts were observed in rat and mouse primary hepatocytes following 1 h exposure to [ring-3H]-N-OH-AAF (0.1-20 microM) and [ring-3H]-N-OAc-AAF (5-20 microM). The patterns of DNA adducts in mouse and rat primary hepatocytes exposed to N-OH-AAF and N-OAc-AF were similar to those obtained in liver following in vivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon (10(-4) M) completely inhibited DNA damage induced by N-OH-AAF in mouse and partially in rat hepatocytes while DNA damage caused by N-OAc-AAF was only partially inhibited by paraoxon (10(-4) M) in both species. Parallel experiments showed that paraoxon, at low concentration (10(-6) M), did not alter either the level of DNA binding or the pattern of adduct formation in rat hepatocytes treated with N-OH-AAF (20 microM). However, at 10(-4) M paraoxon partially blocked DNA binding (60%) and the formation of Gua-C8-AAF (95%) and Gua-N2-AAF (80%) while Gua-C8-AF was increased two-fold. In mouse hepatocytes paraoxon pretreatment (10(-4) M) inhibited the formation of Gua-C8-AF by 70% following exposure to N-OH-AAF (20 microM). Gua-C8-AAF and Gua-N2-AAF were also inhibited but only at 10(-4) M paraoxon.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino-terminal sequence analysis of six cytochrome P-450 isozymes purified by monoclonal antibody directed immunopurification.

Six hepatic microsomal cytochromes P-450 were isolated from 3-methylcholanthrene induced animals by immunopurification using two monoclonal antibodies. Two forms of cytochromes P-450 (MW 56K and 57K) were from Sprague-Dawley rats, two from C57BL/6 mice (56K and 57K), one form from DBA/2 mice (56K) and one form from guinea pigs (53K). NH2-terminal sequences of the first ten amino acids of these cytochromes P-450 were determined by automated Edman degradation. The 56K polypeptides from rats, C57BL/6 mice, and DBA/2 mice were shown to have identical NH2-terminal sequences. The 57K polypeptides from rats and C57BL/6 mice are homologous to each other but exhibit no homology to 56K polypeptides. The 53K polypeptide from guinea pigs has a unique NH2-terminal sequence with no apparent homology to the other five cytochromes P-450.