Biomedical subjects

M Ouzzine

Publications and source records attributed to M Ouzzine.

At least 19 recordsLinked to original sources

PubMed2006

Role of the carboxyl terminal stop transfer sequence of UGT1A6 membrane protein in ER targeting and translocation of upstream lumenal domain.

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.

5' Flanking Region↗

PubMed2004

Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair.

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

Animals↗

PubMed2001

Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes.

OBJECTIVE: To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. METHODS: Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. RESULTS: IL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine. CONCLUSION: The results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Animals↗

PubMed2000

Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system.

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.

Animals↗

PubMed2000

Structure/function of the human Ga1beta1,3-glucuronosyltransferase. Dimerization and functional activity are mediated by two crucial cysteine residues.

Galbeta1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosaminoglycan-protein linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans. In order to gain insight into the structure/function of the enzyme, the human recombinant GlcAT-I was successfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa. Analysis of the electrophoretic mobility of the membrane-bound protein in nonreducing and reducing conditions, together with cross-linking studies, indicated that the membrane-bound GlcAT-I formed active disulfide-linked dimers. GlcAT-I expressed without the predicted N-terminal cytoplasmic tail or secreted as a polypeptide lacking the cytoplasmic tail and transmembrane domain was similarly organized as dimers, suggesting that the structural determinants for the dimerization state are localized in the luminal domain of the protein. In addition, the role of Cys(33) and Cys(301) in that process was investigated by site-directed mutagenesis combined with chemical modification of GlcAT-I by N-phenylmaleimide. Replacement of Cys(33) with alanine abolished the formation of dimers with a concomitant decrease in the catalytic efficiency mainly due to a decrease in apparent maximal velocity and in affinity for UDP-glucuronic acid. On the other hand, N-phenylmaleimide treatment or alanine substitution of the Cys(301) residue inactivated the enzyme. Our study demonstrates that GlcAT-I is organized as a homodimer as a result of disulfide bond formation mediated by Cys(33) localized in the stem region, whereas the residue Cys(301) localized in a conserved C-terminal domain is strictly required for the functional integrity of the enzyme.

Amino Acid Sequence↗

PubMed2000

Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells.

Treatment of Caco-2 cells with the antioxidants quercetin or t-butylhydroquinone led to induced protein levels of UDP-glucuronosyltransferase UGT1A6 (ca. 3-fold over controls) and of the apical conjugate export pump multidrug resistance protein 2 (MRP2; 1.9-fold over controls). In contrast to UGT1A6, MRP2 (symbol ABCC2) was not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Immunocytochemistry demonstrated that MRP2 was only expressed at the brush border domain of Caco-2 cell monolayers. The results indicate that UGT1A6 and MRP2 are coordinately induced by antioxidants, facilitating chemoprotection against phenolic toxins and excretion of conjugates into the intestinal lumen.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2000

Importance of histidine residues for the function of the human liver UDP-glucuronosyltransferase UGT1A6: evidence for the catalytic role of histidine 370.

The human UDP-glucuronosyltransferase isoform UGT1A6 catalyzes the nucleophilic attack of phenolic xenobiotics on glucuronic acid, leading to the formation of water-soluble glucuronides. Based on the irreversible inhibition of the enzyme activity by the histidyl-selective reagent diethyl pyrocarbonate (DEPC), histidine was suggested to play a key role in the glucuronidation reaction. Therefore, the role of four strictly conserved histidine residues (His38, His361, His370, and His485) in the glucuronidation of 4-methylumbelliferone, as reporter substrate, was examined using site-directed mutagenesis. For this purpose, stable heterologous expression of wild-type and mutant UGT1A6 was achieved in the yeast Pichia pastoris. Replacement of histidine residues by alanine or glutamine led to fully inactive H38A, H38Q, and H485A mutants. Substitution of His361 by alanine affected the interaction of the enzyme with the cosubstrate, as indicated by a 4-fold increase in the K(m) value toward UDP-glucuronic acid. Interestingly, H370A mutant presented a severely impaired catalytic efficiency (with a V(max) value approximately 5% that of the wild-type), whereas conservative substitution of His370 by glutamine (H370Q) led to a significant restoration of activity. Whereas H361A was inactivated by DEPC as the wild-type enzyme, this chemical reagent only produced a minor effect on either H370Q or H370A mutant, providing evidence that His370 is probably the reactive histidine residue targeted by DEPC. The dramatic changes in catalytic efficiency on substitution of His370 by alanine and the ability of glutamine to function in place of histidine along with a weak sensitivity of these mutants to DEPC strongly suggest that His370 plays a catalytic role in the glucuronidation reaction.

Alanine↗

PubMed1999

An internal signal sequence mediates the targeting and retention of the human UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum.

The human UDP-glucuronosyltransferase isoform UGT1A6 is predicted to be a type I transmembrane protein anchored in the endoplasmic reticulum by a single C-terminal transmembrane domain, followed by a short cytoplasmic tail. This topology is thought to be established through the sequential action of a cleavable N-terminal signal peptide and of a C-terminal stop transfer/anchor sequence. We found that the deletion of the signal peptide did not prevent membrane targeting and insertion of this protein expressed in an in vitro transcription/translation system or in yeast Pichia pastoris. Interestingly, the same results were obtained when the protein was depleted of both the signal peptide and the C-terminal transmembrane domain/cytoplasmic tail sequences, suggesting the presence of an internal topogenic element able to translocate and retain UGT1A6 in the endoplasmic reticulum membrane in vitro and in yeast cells. To identify such a sequence, the insertion of several N-terminal deletion mutants of UGT1A6 into microsomal membranes was investigated in vitro. The data clearly showed that the deletion of the N-terminal end did not affect endoplasmic reticulum targeting and retention until residues 140-240 were deleted. The signal-like activity of the 140-240 region was demonstrated by the ability of this segment to confer endoplasmic reticulum residency to the cytosolic green fluorescent protein expressed in mammalian cells. Finally, we show that this novel topogenic sequence can posttranslationally mediate the translocation of UGT1A6. This study provides the first evidence that the membrane assembly of the human UGT1A6 involves an internal signal retention sequence.

Biological Transport↗

PubMed1999

Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.

Animals↗

PubMed1997

Arginine 52 and histidine 54 located in a conserved amino-terminal hydrophobic region (LX2-R52-G-H54-X3-V-L) are important amino acids for the functional and structural integrity of the human liver UDP-glucuronosyltransferase UGT1*6.

The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT1*6 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT1*6 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and the Vmax value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT1*6 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT1*6, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1*6. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6.

Animals↗

PubMed1996

Expression of active, human lysyl oxidase in Escherichia coli.

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.

Amino Acid Sequence↗

PubMed1994

Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.

Amino Acid Sequence↗

PubMed1994

Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.

Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.

Affinity Labels↗

PubMed1994

Expression and role of the human liver UDP-glucuronosyltransferase UGT1*6 analyzed by specific antibodies raised against a hybrid protein produced in Escherichia coli.

Characterization of human UDP-glucuronyltransferases (UGTs) has been limited by the unavailability of probes selective for each of several highly related isoforms. To better understand the role of this superfamily in the metabolism of drugs and xenobiotics, we describe a molecular/immunological strategy for discriminating the implication of each human isoenzyme in this process. Specific polyclonal antibodies were generated against the divergent amino-terminal domain of the UGT isoform UGT1*6 which is involved in the detoxification of nucleophilic compounds related to phenols and naphthols in human liver. The novel approach consists of the expression of a N-terminal UGT polypeptide fused to Staphylococcus aureus protein A in Escherichia coli and a single step purification of the fusion protein by immunoaffinity chromatography. Immunoblot and immunoinhibition analysis showed that the antibodies raised against the fusion protein selectively recognized both the denaturated and the native forms of UGT1*6, when expressed in V79 cell lines, but not three other recombinant UGT isoenzymes. In human liver microsomes, specific immunoinhibition analysis demonstrated that glucuronidation by UGT1*6 represented 20 to 50% of the total 1-naphthol UGT activity with a good correlation with the amount of protein selectively quantified on immunoblot. The specific expression of UGT1*6 was found to be significantly reduced in tumoral tissues but enhanced in cholestatic livers, when compared with healthy hepatic tissues. Interestingly, in human kidney microsomes, antibodies revealed a high level of UGT1*6 expression on immunoblot and inhibited 1-naphthol glucuronidation up to 55%, indicating that this isoform is also expressed in kidney and extensively contributes to phenol glucuronidation in this tissue.

Adult↗

PubMed1994

Drug metabolizing enzymes related to laboratory medicine: cytochromes P-450 and UDP-glucuronosyltransferases.

Many studies on drug metabolism have been carried out during the last decades using protein purification, molecular cloning techniques and analysis of polymorphisms at phenotype and genotype levels. These researchers led to a better understanding of the role of drug metabolizing enzymes in the biotransformation of drugs, pollutants or foreign compounds and of their use in laboratory medicine. The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reaction (phase I reactions), which implicate lipophilic compounds. These molecules are modified via monooxygenation, dealkylation, reduction, aromatization, hydrolysis and can be substrates for the phase II reactions, often called conjugation reactions as they conjugate a functional group with a polar, endogenous compound. This review, devoted to cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT), describes essentially the genetic polymorphisms found in humans, their clinical consequences and the methods to assess the phenotypes or genotypes, with a view to studying the interindividual differences in drug monooxygenation and drug glucuronidation. Variations in drug glucuronidation reported here focused essentially on variations due to physiological factors, induction, drug interactions and genetic factors in disorders such as Gilbert's Syndrome and Crigler-Najjar type I and II diseases.

Cytochrome P-450 Enzyme System↗

PubMed1993

Determination of the human liver UDP-glucuronosyltransferase 2B4 domains involved in the binding of UDP-glucuronic acid using photoaffinity labeling of fusion proteins.

The interactions between UDP-glucuronic acid and two human liver UDP-glucuronosyltransferase 2B4 peptides (14-150 and 299-446) purified from E. coli as Staphylococcus aureus protein A fusion proteins have been investigated. Photoaffinity labeling with azidonucleotides ([beta-32P]5N3UDP-Glucuronic acid and [beta-32P]5N3UDP-Glucose) and competition experiments with UDP-glucuronic acid and structurally related compounds emphasized the presence of a specific UDP binding site between amino acids 299 and 446. Moreover, competition experiments strongly suggested an interaction between the amino terminal part of the protein and glucuronic acid. It would involve an electrostatic bond in the binding of the cosubstrate via the carboxyl group of UDP-glucuronic acid and a positively charged amino acid of the N-terminal domain of the enzyme.

Affinity Labels↗

PubMed1993

Glucuronidation of hyodeoxycholic acid in human liver. Evidence for a selective role of UDP-glucuronosyltransferase 2B4.

Monospecific polyclonal antibodies were raised against a variable amino-terminal domain (amino acids 14-150) of a human liver form of UDP-glucuronosyl-transferase conjugating bile acids, UGT2B4 (Jackson, M. R., McCarthy, L. R., Harding, D., Wilson, S., Coughtrie, M. W., and Burchell, B. (1987) Biochem. J. 242, 581-588), expressed as a fusion protein in Escherichia coli. The antibodies were able to recognize the protein, stably expressed in a genetically engineered eukaryotic V79 cell line, against which they were directed. The specificity of these antibodies allowed their use for analyzing the substrate specificity of this isoform in human liver, as well as for determining its contribution to the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic acid. Western blot analysis of microsomal proteins demonstrated the presence of UGT2B4 exclusively in human liver and not in human kidney. In human liver microsomes, the antibodies were able to inhibit and precipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidation activity, but had no effect on activities toward several other substrates, such as phenols, bilirubin, or other bile acids, especially hyocholic acid and the steroids 4-hydroxyesterone and estriol. Moreover, Western blot analysis and immunoinhibition studies of human liver microsomes from healthy patients and from patients presenting liver diseases revealed a good correlation between the glucuronidation rate of hyodeoxycholic acid and the UGT2B4 expression level. The absence of immunoinhibition of hyodeoxycholic acid conjugation with UDP sugars other than UDP-glucuronic acid suggests the involvement of different enzymatic systems in the glucosidation and xylosylation of hyodeoxycholic acid. Altogether, the results provided strong evidence for the specific and predominant involvement of UGT2B4 in the 6-O-glucuronidation of this bile acid via a UDP-glucuronic acid-dependent mechanism.

Animals↗

PubMed1993

[What has the reform of primary care meant in the community approach to arterial hypertension?].

OBJECTIVE: To evaluate an arterial Hypertension programme (AHP), with analysis of its feasibility and the problems inherent in the methodology used. DESIGN: A retrospective evaluation of the results of an AHP programme. A population projection of the results by means of an extrapolated calculation. SETTING: An urban Health Centre. PATIENTS: A random sample of 282 patients selected from the total of 564 individuals diagnosed with AHT over the 6 years of the programme's operation. MEASUREMENTS AND MAIN RESULTS: The 564 patients in the records made up 16.5% of the Health Area's adult population. The iceberg phenomenon (the percentage of undiagnosed hypertension sufferers) has evolved from 59% in 1988 to the present 17%. 7.4% of the sample received no treatment. Average AHT figures for the patients in treatment were 143 +/- 15 mm Hg systolic pressure and 85 +/- 9 mm Hg diastolic. 75% had adequate control of their tension figures (< 160/95). The population projection of these findings indicated that 57% of Hypertension sufferers calculated to be living in the Health area were diagnosed and monitored. CONCLUSIONS: The results show that the AHT situation among the population has substantially improved. The methodology used is easy to manage and can be of service in the evaluation of the impact of AHT programmes on the population.

Adult↗