Human Tissue Homogenates Research Articles

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50–80 μl of the aqueous layer onto a C 18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer—acetonitrile—methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.

Human hepatic N-acetylglutamate content and N-acetylglutamate synthase activity. Determination by stable isotope dilution

N-Acetyl-L-glutamate (N-acetylglutamate) content and N-acetylglutamate synthase activity ranges were established in human liver tissue homogenates by stable isotope dilution. The methods employ N-[methyl-2H3]acetyl[15N]glutamate as internal standard, extraction of N-acetylglutamate by anion-exchange technique and its determination by g.l.c.-mass spectrometry by using selected ion monitoring. Hepatic N-acetylglutamate content in 16 different human livers, normal in structure and function, ranged from 6.8 to 59.7 nmol/g wet wt. (25.0 +/- 13.4 mean +/- S.D.) or from 64.6 to 497.6 nmol/g of protein (223.2 +/- 104.2 mean +/- S.D.). In vitro, N-acetylglutamate synthase activity in liver tissue homogenate ranged from 44.5 to 374.5 (132.0 +/- 90.6 mean +/- S.D.) nmol/min per g wet wt. or from 491.7 to 3416.9 (1159.6 +/- 751.1 mean +/- S.D.) nmol/min per g of protein. No correlation was found between hepatic N-acetylglutamate concentrations and the respective maximal enzymic activities in vitro of N-acetylglutamate synthase. The marked variability in this system among individual livers may reflect its regulatory role in ureagenesis.

Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticulocytes and tumors

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism, but little is known about its physiological functions. We purified dThdPase from human placenta and used it for antibody preparation. The purified material appears as a single band at 55 000 dalton on sodium dodecylsulfate-polyacrylamide gel electrophoresis. We obtained a specific antibody raised in rabbits that detected a single polypeptide with a molecular weight of 55 000 dalton in the post nuclear homogenates of several human tissues, on immunoblotting. Using the same technique, dThdPase was highly expressed in the liver, lung, spleen, lymph nodes and peripheral lymphocytes. Immunohistochemical staining revealed that macrophage-like cells contained a much higher amount of dThdPase than parenchymal cells in the liver and lung. dThdPase was found to be highly expressed in T- and B-cell-type malignant lymphoma cells, but low in lymphoblastic and myeloblastic leukemia cells. We also found that carcinomas in the stomach, colon and ovary contained higher amounts of this enzyme than non-neoplastic regions of the tissues. These data suggest that dThdPase plays a role in proliferation and/or differentiation of leukocytes and in cancer proliferation.

N-Acetylglutamate Content in Liver and Gut of Normal and Fasted Mice, Normal Human Livers, and Livers of Individuals with Carbamyl Phosphate Synthetase or Ornithine Transcarbamylase Deficiency

N-acetylglutamate (NAG) content was measured in homogenates of liver and small intestine obtained from normal and 24-h starved syngeneic mice. Subsequently, NAG was determined in normal, and in carbamyl phosphate synthetase I and ornithine transcarbamylase enzyme-deficient human liver tissue homogenates. The method used in this study, which is direct and highly specific, used anion exchange extraction, gas chromatographic separation, and mass spectrometric detection and quantitation. Hepatic NAG content in the fed animals was 94.8 +/- 19.8 nmol/g tissue or 602.5 +/- 168.4 nmol/g protein (mean +/- SD, n = 5), whereas it was much lower in the fasted mice (49.4 +/- 13.0 nmol/g tissue or 330.1 +/- 113.9 nmol/g protein, mean +/- SD, n = 5). The magnitude of the difference was much smaller for intestinal NAG content, 19.8 +/- 5.4 nmol/g tissue or 205.3 +/- 70.3 nmol/g protein (mean +/- SD, n = 5) in the fed mice and 14.2 +/- 4.3 nmol/g tissue or 168.1 +/- 80.8 nmol/g protein (mean +/- SD, n = 5) in the fasted mice. The concentrations of hepatic NAG in normal human livers (controls) ranged from 19.3 to 67.1 nmol/g tissue (41.6 +/- 19.3, mean +/- SD, n = 5) or from 193 to 764.3 nmol/g of protein (437.5 +/- 233.4, mean +/- SD, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Neuromelanin synthesis in rat and human substantia nigra

A relation between neuromelanin synthesis and vulnerability of dopaminergic neurons is suggested by the fact that heavily pigmented cells are preferentially lost in aging and Parkinson's disease and that the dopaminergic neurotoxin MPP+ (1-methyl-4-phenyl-pyridine) binds to neuromelanin. To elucidate the mechanism of neuromelanin synthesis, we studied the formation of melanin in homogenates of human and rat substantia nigra tissue "in vitro". It was found that enzymatic processes accounted for 70% and 90% of the melanin formation in homogenates of human and rat tissue, respectively. The enzymatic synthesis was due to the activity of monoamine oxidase (MAO), since it was prevented by selective inhibitors of this enzyme. Both MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and MPP+ inhibited melanin formation, probably due to their ability to inhibit MAO. No evidence was found for involvement of cytochrome P-450 monooxigenases, which have been postulated to exist in central catecholaminergic neurons. Proadifen reduced melanin formation, not necessarily because it is an inhibitor of P-450 monooxigenases, but rather as it is also a potent inhibitor of MAO. Some antioxidants like ascorbic acid, but not agents destroying hydrogen peroxide, inhibited melanin formation. The findings suggest that the formation of neuromelanin in the substantia nigra involves MAO and non-enzymatic oxidative processes.

17 β-Hydroxysteroid oxidoreductases of human fetal and adult tissues: Immunological cross-reactivity with an anti-human placental cytosolic 17 β-hydroxysteroid oxidoreductase antibody

To determine whether the immunological determinants of human placental 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) were present in 17 beta-HSORs of tissues of the human fetus and adult and of various non-human cells maintained in culture, western immunoblot analysis was conducted by use of a polyclonal antibody directed against determinants of the placental cytosolic enzyme. Tissues and cells were evaluated for the presence of immunocross-reactive proteins with a relative molecular mass (Mr) similar to that of placental 17 beta-HSOR (approximately 34,000). By use of homogenates of human fetal tissues, immunostaining of 17 beta-HSORs of Mr approximately 34 kDa was detected in trophoblast, fetal adrenal neocortex, fetal zone of the adrenal gland, liver, intestine, kidney, brain, lung, skin, heart, spleen, pancreas, chorion laeve, and, occasionally, amnion. Immunostaining at Mr approximately 34 kDa also was demonstrated by use of cytosolic preparations of fetal tissues and, in some cases, by use of unwashed microsomal fractions; this protein was either absent or present in almost undetectable amounts in washed microsomes, except for placenta and fetal brain. Immunostaining at approximately 34 kDa was demonstrated occasionally in decidua of pregnant women by use of homogenates, but was not detected in endometrium or myometrium of non-pregnant women, testis of an adult man, mouse and rat Leydig tumour cells, mouse and rat adrenal tumour cells, and normal bovine adrenocortical cells.

Identification and characterisation of 5-hydroxytryptamine 3 recognition sites in human brain tissue.

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.

Detection of a novel serotonin receptor subtype (5-HT1E) in human brain: interaction with a GTP-binding protein.

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)

μ Opiate receptors are selectively labelled by [ 3H]carfentanil in human and rat brain

[ 11C]Carfentanil is a potent opioid agonist currently in use as a specific PET (position emission tomography) scan radioligand for brain μ opioid receptors. In order to investigate the receptor interactions of carfentanil in detail [ 3H]carfentanil was used as a radioligand for labelling receptors in rat and human brain tissue homogenates. [ 3H]Carfentanil was found to bind saturably and with high affinity (K D = 0.08 ± 0.01 nM) to membranes prepared from human cortical (B max = 42 ± 3 fmol/mg) and thalamic (B max = 84 ± 3 fmol/mg) tissues and rat cortex (B max = 82 ± 4 fmol/mg) and deincephalon (B max = 105 ± 5 fmol/mg). Association (1.23 ± 0.19 × 10 10Mol −1 × min −1 and dissociation rate (0.19 ± 0.03 min −1) constants were determined in human cortical tissues; results from studies in rat cortical, and rat diencephalon tissue homogenates produced similar kinetic rate constants. Competition studies with a variety of drugs indicated that [ 3H]carfentanil interacts primarily with μ opioid receptors in the four tissues studied; the affinities of a series of non-radioactive opioid ligands were essentially identical in the four tissues (correlation coefficients = 0.88−0.93). Naloxone, morphine, DAGO ([D-Ala 2-MePhe 4-Gly-ol 5]enkephalin), DADL (D-Ala 2-D- Leu 5]enkaphalin) and EKC (ethylketazocine) potently displaced specific [ 3H]carfentanil binding with nM potency while the κ agonist U-69593, the σ agonists (+)-SKF 10047, (+)-3-PPP ((3-hydroxyphenyl)-N-propylpiperidine) and haloperidol and PCP (phencyclidine) were less potent displacing agents. The higher affinities of DAGO and morphine versus DADL for the [ 3H]carfentanil binding site indicates that δ opioid receptors are not being labelled. These data indicate that [ 3H]carfentanil is a high affinity, specific μ opioid receptor radioligand that may be of use in vitro for studying μ opioid receptors and supports the PET scanning data indicating the μ opioid receptor specificity of [ 11C]carfentanil. The molecular kinetic rate constants determined in vitro will be of assistance in using modelling procedures to interpret PET scanning information.

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu 13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30°C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl 2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu 13]motilin or synthetic natural porcine motilin indicated a dissociation constant ( K d) of 3.6 ± 1.6 nM and a maximal binding capacity ( B max) of 77 ± 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent K d of 2.2 ± 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 ± 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.

A Monoclonal Antibody Against Human Testicular Anti‐Müllerian Hormone

Using immunochromatography on a polyclonal antibody, testicular anti-Müllerian hormone (AMH) was purified from homogenates of human fetal testicular tissue and used as an antigen for hybridoma production. Two IgM clones were obtained. Both recognized AMH on biopsies of human testicular tissue and one blocked its anti-Müllerian activity. This monoclonal antibody (MAb) exhibited a relatively high affinity for AMH, and was studied further. It is interspecific, recognizing AMH in other mammalian species. The study of this MAb in relation to an IgM MAb raised against bovine AMH (bAMH) indicates that both MAbs recognize different but related epitopes on bAMH.

A radioisotopic assay of picomolar concentrations of coenzyme A in liver tissue

A single-step enzyme assay using [ 14C]palmitic acid and bacterial acyl-coenzyme A synthetase (EC 6.2.1.3) is described for the determination of reduced coenzyme A (CoASH) levels in liver samples. Use of this technique provides a rapid and accurate determination of CoASH in the range 1–250 pmol. Application of the method to the quantitation of CoASH in samples of human liver tissue and rat liver homogenate, isolated hepatocytes, and mitochondria is described.

Prostatic growth factor: purification and structural relationship to basic fibroblast growth factor.

Prostatic growth factor (PrGF) was purified from alkaline homogenates of human benign prostatic hyperplastic tissue by a combination of ammonium sulfate precipitation, heparin affinity chromatography, and cation-exchange chromatography. The 17,600-dalton, basic (pI 10.2) PrGF is related to basic fibroblast growth factor (bFGF) since antisera raised against synthetic peptides with sequence homologies corresponding to an internal peptide and amino- and carboxyl-terminal peptides of bFGF react with the growth factor. The growth factor appears larger than bFGF, suggesting that additional amino-terminal sequences may be present as a result of alkaline extraction in the presence of protease inhibitors.

Prostacyclin production by cultured human endothelial cells and tissue homogenates in response to fish liver extracts

Conference Article| June 01 1987 Prostacyclin production by cultured human endothelial cells and tissue homogenates in response to fish liver extracts C. EVANS; C. EVANS 1Department of Chemistry and Biochemistry, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. BILLINGTON; D. BILLINGTON 1Department of Chemistry and Biochemistry, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K. Search for other works by this author on: This Site PubMed Google Scholar F. A. McEVOY F. A. McEVOY 1Department of Chemistry and Biochemistry, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (3): 563–564. https://doi.org/10.1042/bst0150563 Article history Received: December 22 1986 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation C. EVANS, D. BILLINGTON, F. A. McEVOY; Prostacyclin production by cultured human endothelial cells and tissue homogenates in response to fish liver extracts. Biochem Soc Trans 1 June 1987; 15 (3): 563–564. doi: https://doi.org/10.1042/bst0150563 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. I: Validation of methods.

The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.

Peroxisomal fatty acid oxidation in rat and human tissues. Effect of nutritional state, clofibrate treatment and postnatal development in the rat

Oxidation of palmitate 14C-labeled in different positions was assayed in the absence and presence of antimycin and rotenone in homogenates of various rat and human tissues to determine total and peroxisomal oxidation and acetyl group production. Total and antimycin-insensitive palmitate oxidation rates were higher in rat heart, liver and quadriceps muscle than in the corresponding human tissues. The proportion of antimycin-insensitive oxidation of [1- 14C]palmitate was 17–35% in tissues of starved rats and in human muscles and fibroblasts, but peroxisomal production of acetyl groups amounted only to 5–11% of that by mitochondria. The mean number of peroxisomal β-oxidation cycles was 1.5–2.5 per palmitate molecule. The nutritional state markedly influenced the total oxidation rate and the antimycin-insensitive proportion in rat liver. Clofibrate feeding increased total and antimycin-insensitive oxidation rates in liver, heart and kidney, but not in quadriceps muscle. Total oxidation capacity was maximal in rat liver at weaning, and in rat heart at an age of 70 days. Antimycin-insensitive oxidation rates increased in rat liver and heart at postnatal development up to weaning. A marked proportion of lignocerate oxidation was antimycin-sensitive in rat tissues.

Species-dependent differences of the biochemical properties of diamine oxidase

1. 1. Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at −20°C. 2. 2. The K m -value for putrescine was 90 μM in mouse kidney or intestine. In rats different K m -values were observed before (272 μM) and after freezing (102 μM). 3. 3. A similar effect was observed with DAO in human kidney (321 and 39 μM, respectively). 4. 4. Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. 5. 5. The enzyme remaining in the intestine showed the lower K m -value.

Specific binding of gonadotrophin-releasing hormone and an agonist to human corpus luteum homogenates: characterization, properties, and luteal phase levels.

The binding of radiolabeled GnRH and GnRH agonist (GnRHA) was studied in homogenates of human luteal tissue using a polyethylene glycol precipitation technique. GnRHA binding to human luteal homogenates was dependent on pH, was inhibited by a number of divalent metal ions, and was of moderately high affinity (Ka = 3 X 10(7) M-1). Binding of both radiolabeled GnRH and GnRHA increased linearly with increasing homogenate concentration, and binding of either tracer was parallel with different corpus luteum (CL) homogenates. Moreover, similar concentrations of unlabeled GnRH or GnRHA blocked the binding of either tracer, indicating that both hormones bound to a common site. The rate of dissociation of bound ligand was very low at 0 C, but was extremely rapid at physiological temperatures (t1/2 = 2.5-3.5 min). Human luteal GnRHA-binding sites were partially solubilized by treatment with Triton X-100 and were sensitive to trypsin and pronase, but not hyaluronidase. GnRHA binding to homogenates of human CL obtained at each stage of the luteal phase varied markedly, with levels ranging from less than 20 to greater than 1600 pg/mg DNA for CL from the (midluteal) stage of the menstrual cycle. This variability could not be accounted for by assay irreproducibility, loss of binding activity during storage, or occupancy of binding sites by endogenous ligand. These observations may help to explain negative reports from other groups, which demonstrated little or no specific binding of GnRH to human luteal tissue and no effect of GnRH on steroidogenesis using dispersed human luteal cells in vitro. We conclude that human luteal homogenates possess specific, moderately high affinity binding sites for GnRH and its agonist analogs.

Cathepsin D, L and B in inflamed human gingiva.

Cathepsin D, cathepsin L, and cathepsin B activities were detected in homogenates of human gingival tissue from 20 patients with different degrees of periodontal disease and in one patient with juvenile periodontitis. Clinical plaque index (PI), gingival index (GI), and pocket depth were determined. Patients were divided into three groups with regard to the severity of the disease. Averaged values for the activities of all three lysosomal proteinases increased from the group with less damaged tissue to the group with more advanced periodontal disease. When the correlations between the specific activities of the cathepsins and pocket depth in each of 20 patients were considered by the linear regression analysis, the correlation coefficient for cathepsin D was much higher than for the other two cysteine proteinases. Cathepsin D was also extracted from the homogenates by specific immuno‐adsorbtion on anticathepsin ‐ D antibody ‐ Sepharose and quantitatively determined; the average amount of enzyme ranged from 118.5 to 184.5 μg per mg of total protein extracted from the tissues and was positively correlated to pocket depths.

Distribution of angiotensin converting enzyme in human tissues

Angiotensin converting enzyme (EC 3.4.15.1, dipeptidyl carboxypeptidase, ACE, Kininase II), the peptidase which transforms inactive decapeptide, angiotensin I, to the pressor octapeptide, angiotensin II, and which catalyses also the degradation of vasodilative nonapeptide bradykinin, was measured in 27 human tissue homogenates and physiological fluids. Two assays were used: one which measures the hydrolysis of the substrate hippuryl-glycyl-glycine, by means of high performance liquid chromatography and another, using a colorimetric assay measuring the cleaved glycyl-glycine after arylation with picrylsulfonic acid. All the tissues studied contained measurable converting enzyme activities which were inhibited by captopril (SQ 14.225) in low concentrations. High specific activities of converting enzyme were found in several tissues of the intestinal and urogenital tract, but the highest activity was found in benign prostatic hyperplasia. Normal prostate and prostatic adenocarcinoma have a much lower activity. Results obtained for human tissues are compared with those found in animals.