Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study.

BACKGROUND: Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. RESULTS: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1). CONCLUSIONS: The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.

Elastase secretion by stimulated macrophages. Characterization and regulation.

Thioglycolate-stimulated mouse peritoneal macrophages secrete a Proteinase which degrades insoluble elastin. There is little elastase activity in cell lysates but the bulk of the enzyme accumulates extracellularly during culture in serum-free medium. The secretion of elastase is sustained for over 12 days in culture and continued secretion of elastase requires protein synthesis. Unstimulated macrophages secrete very little elastase activity but can be triggered to secrete higher levels of this enzyme by phagocytosis and intracellular storage of latex particles. The macrophages elastase is a distinctive proteinase differing from the elastases of pancreas and granulocytes and is distinct from the other secreted proteinases of macrophages, namely, collagenase and plasminogen activator. The macrophages elastase is a serine proteinase and is inhibited by di-isopropyl phosphoro-fluoridate, ovoinhibitor, EDTA, dithiothretiol, and serum. Its activity is little affected by soybean trypsin inhibitor, turkey ovomucoid and chloromethyl ketones derived from tosyl lysine, tosyl phenylalanine, and acetyltetra alanine. Hydrolysis by macrophage elastase of chromogenic ester substrates for pancreatic elastase could not be detected. Elastase secretion by stimulated macrophages exceeds that by primary and established fibroblast cell strains. It is likely that elastase secretion by macrophages plays a major role in the pathogenesis of chronic destructive pulmonary diseases such as emphysema.

Clinical approach to the patient with chronic hepatitis C infection and normal aminotransferases.

Approximately 30% of patients with chronic HCV infection have persistently normal alanine aminotransferase levels (PNALT). Most of these patients have minimal or mild inflammation and absent or minimal fibrosis, although occasionally cirrhosis and hepatocarcinoma may be seen. Overall, liver histology is significantly less severe than in patients with elevated ALT levels, and most follow-up studies have reported stability of the disease, with minimal fibrosis progression over years, and thus a disease with a favorable prognosis. Nevertheless, a few studies have shown more recently that many patients with PNALT, may have elevations in ALT over time, and almost 20-30% have a significant progression of fibrosis, being eligible for antiviral therapy. During the last decade it has been demonstrated that in chronic HCV infection with PNALT, combination antiviral therapy with peg interferon-alpha plus ribavirin is efficacious, safe, and associated with significant improvements in health-related quality of life, and the decision whether to treat or not this patients should be based on multiple factors including: age, HCV genotype, histology, patients motivation and adherence, symptoms and comorbidity, rather than on ALT levels alone.

Salivary acetylcholinesterase as a biomarker for organophosphate exposure.

BACKGROUND: Workers exposed to organophosphate (OP) pesticides are required to undergo periodic statutory medical surveillance in several countries. AIM: To study the relationship between serum, erythrocyte and saliva acetylcholinesterase (AChE) levels and to explore the use of salivary AChE as potential biomarker for OP exposure. METHODS: A cross-sectional study was conducted on 19 healthy adult male lead-exposed workers who were undergoing six monthly statutory medical examination. Passive drool saliva samples were collected from each worker. Each blood sample was tested for serum and erythrocyte AChE, and each saliva sample was tested for AChE. RESULTS: Among the 19 subjects, the mean (+/-standard deviation) of salivary, erythrocyte and serum AChE/cholinesterase were 22.7 (+/-17.4), 17171 (+/-1467), 8861 (+/-1876) U/l, respectively. There was a moderate correlation between salivary and erythrocyte AChE (r = 0.42, P = 0.071), but not salivary and serum AChE (r = -0.17, P = 0.48). The level of AChE in saliva was approximately 1820 times lower than AChE in erythrocytes. CONCLUSION: It is probably not feasible to use saliva as a replacement for blood for the measurement of AChE levels. This is because of the much lower levels of AChE in saliva relative to erythrocytes, the weak correlation between the two measurements and the previously reported high intra-individual variation of salivary AChE.

The Diagnostic Usefullness of the Salivary Pepsin Test in Symptomatic Laryngopharyngeal Reflux.

OBJECTIVE: Laryngopharyngeal Reflux (LPR) is a disease characterized by the presence of symptoms, signs and tissue alterations in the aero-digestive upper tract as a consequence of the gastric contents retrograde movement. In most cases diagnosis is clinical and it is established by the presence of symptoms and endoscopic laryngeal signs. The aim of the study was to determine the sensitivity, specificity, positive and negative Likelihood Ratio (LR) of the salivary pepsin assay (PEP-test, RD Biomed, Hull, UK) as diagnostic tool of LPR. STUDY DESIGN: Diagnostic Accuracy Study. METHOD: 221 subjects aged between 26 and 68 years were recruited. All subjects completed the Reflux Symptom Index scale. PEP-test was carried out on fasting subjects, and a second test was performed one hour after the main meal, only on those subjects with a fasting negative result. RESULTS: Fasting PEP-test showed a 98% specificity, 40% sensitivity, positive LR of 16.4 and negative LR of 0.61. The use of both PEP-test showed a 95% specificity, 48% sensitivity, positive LR of 9.61 and negative LR of 0.55. CONCLUSIONS: The PEP-test is a simple, inexpensive, non-invasive and easily reproducible test that should be considered as an alternative diagnosis tool for LPR diagnosis. When there is a clinical suspicion of LPR disease, a positive result on the test could be considered diagnostic, but on subjects with negative results it should be complemented with more complex tests such as the 24-hour dual-channel pH-metry.

Synthesis and single enzyme activity of a clicked lipase-BSA hetero-dimer.

Click chemistry is used to construct a novel lipase-BSA hetero-dimer, in which the latter protein acts as a foot enabling the anchoring of the enzyme onto the surface for single enzyme studies.

The transmission of anaerobic periodontopathic organisms.

The oral microbial flora is unique, and available evidence indicates that it is passed vertically from parents to children. In this investigation, we used a chairside assay for the N-benzoyl-DL-arginine-2-naphthylamide (BANA)-sensitive enzyme found in Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythensis, to determine the prevalence of these BANA-positive species in young children and their caregivers. We predicted that if the BANA enzyme was found in plaque samples of children, it would also be present in the plaque samples of the caregivers. Forty-four percent of 150 children had at least one plaque sample positive for the BANA enzyme. If the caregiver was BANA-positive, the odds of the child also being BANA-positive was 35 times more than for a child with a BANA-negative caregiver, after adjustment for the child's age and papillary bleeding score (PBS). Other significant predictors were the PBS of children (p < 0.001), a history of periodontal disease, and the ages of the caregivers (p < 0.001).

Relationship between gingival clinical parameters and the reactivity of the BANA test in subgingival samples from children.

Gingivitis is the first manifestation of periodontal disease, and is characterized by painless and slow evolution. Early diagnosis and intervention must be done to avoid the possibility of precocious periodontitis during the childhood or teenage years. The enzymatic BANA test (N-benzoyl-DL-arginine-naphthylamide) was used to evaluate subgingival samples from 54 children between 6 and 9 years of age. Plaque index (PI) and gingival index (GI) were assessed according to the criteria recommended by Löe (1967). Subgingival plaque was collected from the region that featured the greatest periodontal alteration, represented by a higher gingival index. Resulting data were grouped individually according to visible and non-visible plaque and bleeding and non-bleeding gingiva. Results showed that there was no statistically significant correlation between the presence of visible plaque and the positivity of the BANA test, nor was there a statistically significant correlation between the presence of bleeding and the positivity of the BANA test in subgingival samples obtained from children. This study concluded that the BANA test is not an ideal diagnostic test to be applied to children.

The transmission of BANA-positive periodontal bacterial species from caregivers to children.

BACKGROUND: The purpose of the authors' study was to use the N-benzoyl-DL-arginine-2-naphthy-lamide (BANA) test (BANAMet LLC, Ann Arbor, Mich.) to obtain information regarding the prevalence of an enzyme unique to certain periodontal pathogens in plaque samples of children, as well as the potential transmission of these pathogens from caregivers to children. METHODS: The authors tested 218 subjects (3 to 10 years old) and 195 care-givers at four pediatric dentistry clinics in Taipei, Taiwan. RESULTS: Forty-four percent of the children had at least one plaque sample that tested positive and/or weakly positive. Positive results were more frequent in the mixed dentition, as well as in children with gingivitis (P < .001). A logistic regression model showed that if the BANA test results for the care-giver were positive, the odds of the child's also having positive test results were 55 times greater (P < .001; confidence interval [CI] = 14 to 224) than those for a child whose caregiver had negative BANA test results. Other predictors were the presence of a mixed dentition (P < .001; odds ratio [OR] = 11; CI = 3.5 to 33.5) and the children's papillary bleeding scores (P < .001, OR = 3.1, CI = 2.0 to 4.7). CONCLUSION: The BANA test results were positive for almost one-half of the children. A positive reaction was associated with gingivitis, a mixed dentition, a BANA-positive caregiver or a caregiver with a history of periodontal disease in the family. CLINICAL IMPLICATIONS: The authors propose an anaerobic periodontal infection risk model in which children with a mixed dentition who have gingivitis and a caregiver with a history of periodontal disease would undergo the BANA test.

Screening of periodontitis with salivary enzyme tests.

The purpose of this study was to determine the usefulness of salivary biochemical markers for the screening of periodontal disease and examine the agreement between the results of saliva enzyme tests and those of probing depth. The present study included a total of 187 subjects who underwent annual medical check-ups at the Comprehensive Health Care Center, Honjo, Saitama Prefecture, Japan. Periodontal pocket probing was performed with a WHO probe, and various enzymes and biochemical parameters in saliva were measured. For lactate dehydrogenase (LDH), the proportions of the five isoenzymes were calculated. To decide the cut-off point for each enzymatic activity, receiver operating characteristic curves (ROC curves) were constructed and the points of minimum difference between sensitivity and specificity were decided. Among the biochemical markers tested, salivary LDH level had the highest sensitivity and specificity (sensitivity 0.66, specificity 0.67), while salivary levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN) also had sensitivity and specificity above 0.60. Among the LDH isoenzymes, LDH4 and LDH5 dominated in whole saliva samples. Salivary LDH may be a feasible and useful parameter for the screening of periodontal disease, while salivary AST and BUN also appear to be potentially useful for this purpose.

A simple radioimmunoassay of human placental alkaline phosphatase (Regan isoenzyme) using specific antibody polymers.

Rabbit antiserum against highly purified high-molecular-weight B-variant of human placental alkaline phosphatase (M.W. 200,000) was rendered monospecific by absorption with polymerized pooled male serum proteins; the absorbed antiserum was then polymerized with ethyl chloroformate and used in radioimmunoassay as a stable solid-phase immunoabsorbent. Homogeneous preparation of the enzyme, with a specific activity of 477 mumoles phenol per mg per min, was also obtained by absorbing the chromatographically purified enzyme with polymerized rabbit antiserum directed to whole human serum proteins; the pure enzyme was then labeled with 125-I as the tracer retaining at least 80% of its antigenicity. Only a minute quantity of the polymerized antibody particles is required for each assay in admixture with the labeled and unlabeled enzyme. By adding a small amount of starch-gel particles before low-speed centrifugation, complete phase separation was achieved. The radioimmunoassay could detect 0.4 to 0.8 ng enzyme protein per tube, which is comparable to the sensitivity achieved by enzymic assays. However, radioimmunoassay is advantageous over the enzymic assay in being direct, specific (no interference by the nonplacental-type alkaline posphatase), and capable of detecting both catalytically active and inactive forms of the enzyme. Native variants of placental-type alkaline phosphatase including Regan isoenzyme and Nagao isoenzyme (D-phenotype of normal placental alkaline phosphatase), could thus be directly determined by this procedure in the clinical specimens.

A new oxygen transport agent.

Modern highly purified and chemically modified hemoglobin-based oxygen carriers (HBOC) are free of significant side effects on kidneys and coagulation, and they do not possess ABO antigens, allowing transfusion without knowledge of the respective blood group. Even at room air oxygen concentrations HBOC can compensate for intravascular volume deficits in hemorrhagic shock, including restoration of colloid osmotic pressure and organ perfusion, and deliver oxygen to organs and tissues during nearly complete blood exchange. In animal experiments and clinical trials all HBOC showed a vasoconstrictive side-effect which is mainly caused by nitric oxide scavenging, and to a lesser extent by reactive vasoconstriction because of precapillary oxygen off-loading. The study by Bjorkholm in this issue of the journal (see page 505) investigates the application of a moderate dose of the newly designed HBOC, MP4, in volunteers. MP4 has a high molecular size and a very low p50 resulting in a high oxygen affinity thus avoiding significant (pre)capillary oxygen off-loading. No significant rises in blood pressure or major laboratory abnormalities were seen after MP4 infusion. This new HBOC may be applicable in patients as a red blood substitute where vasoconstriction must be avoided. In addition, poststenotic tissue oxygenation might be a further indication. However, the number of treated volunteers and the infused dose of MP4 were both are very small. Therefore, one cannot draw conclusions on the safety, tolerability and efficacy of MP4 in terms of red cell replacement when large amounts of oxygen carriers are needed.

A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol.

BACKGROUND AND OBJECTIVES: MP4 (Hemospan), a hemoglobin-based oxygen carrier, has been designed to deliver oxygen to hypoxic tissues without causing vasoconstriction. A phase I clinical trial of MP4 was undertaken to evaluate whether MP4 elicits the clinical side effects associated with previous hemoglobin-based solutions. DESIGN AND METHODS: Twelve volunteers were studied. One cohort (n=4) received 50 mg/kg of MP4, a second (n=4) received 100 mg/kg of MP4, and the third (n=4) received lactated Ringer's solution. Single blind infusions were given at 5 mL/min. Vital signs and symptoms, hematologic parameters, serum chemistry, renal and electrocardiographic measurements were monitored for 15 days after dosing. RESULTS: Five mild adverse events occurred in the controls and 2 each in the 50 mg/kg and 100 mg/kg MP4 groups. None was severe or judged related to MP4 administration by the principal investigator. There were no clinically significant alterations in blood pressure or heart rate, and there were no gastrointestinal symptoms, abdominal or flank pain, loss of appetite or clinically significant alterations of liver or pancreatic enzymes. In one subject (100 mg/kg of MP4), amylase and lipase were slightly above the upper limit of normal 4 hours after dosing, but without associated symptoms or signs. Pharmacokinetic analysis of plasma hemoglobin (assuming no hemolysis) yielded an estimated half-life (T1/2) of 43 hours in the 100 mg/kg MP4 subjects. INTERPRETATION AND CONCLUSIONS: MP4 appears to have a favorable safety profile. Subjects in both study groups survived and did no less well than those in the control group.

Impact of tracheal cuff shape on microaspiration of gastric contents in intubated critically ill patients: study protocol for a randomized controlled trial.

BACKGROUND: Ventilator-associated pneumonia (VAP) is the most common infection in intubated critically ill patients. Microaspiration of the contaminated gastric and oropharyngeal secretions is the main mechanism involved in the pathophysiology of VAP. Tracheal cuff plays an important role in stopping the progression of contaminated secretions into the lower respiratory tract. Previous in vitro studies suggested that conical cuff shape might be helpful in improving tracheal sealing. However, clinical studies found conflicting results. The aim of this study is to determine the impact of conical tracheal cuff shape on the microaspiration of gastric contents in critically ill patients. METHODS/DESIGN: This prospective cluster randomized controlled crossover open-label trial is currently being conducted in ten French intensive care units (ICUs). Patients are allocated to intubation with a polyvinyl chloride (PVC) standard (barrel)-shaped or a PVC conical-shaped tracheal tube. The primary objective is to determine the impact of the conical shaped tracheal cuff on abundant microaspiration of gastric contents. Secondary outcomes include the incidence of microaspiration of oropharyngeal secretions, tracheobronchial colonization, VAP and ventilator-associated events. Abundant microaspiration is defined as the presence of pepsin at significant level (>200 ng/ml) in at least 30 % of the tracheal aspirates. Pepsin and amylase are quantitatively measured in all tracheal aspirates during the 48 h following inclusion. Quantitative tracheal aspirate culture is performed at inclusion and twice weekly. We plan to recruit 312 patients in the participating ICUs. DISCUSSION: BEST Cuff is the first randomized controlled study evaluating the impact of PVC tracheal-cuff shape on gastric microaspirations in patients receiving invasive mechanical ventilation. Enrollment began in June 2014 and is expected to end in October 2015. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01948635 (registered 31 August 2013).

Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF.

A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.

Collagenolytic enzymes in gingival crevicular fluid as diagnostic indicators of periodontitis.

Periodontal diseases are associated with the production of several families of enzymes that are detectable in gingival crevicular fluid and that are released by stromal, epithelial or inflammatory cells. Measurement of collagen degrading enzymes in crevicular fluid could contribute to insights into pathogenesis of periodontal diseases and also provide a rational basis for the development of novel diagnostic tests. However, similar to the development of other diagnostic tests, the appropriate validation of collagenolytic enzymes as diagnostic indicators is dependent on clearcut demonstrations of the identity of the enzyme in the assay, and the reproducibility, diagnostic accuracy, and clinical utility of the test. If collagenolytic enzymes are to be of clinical usefulness, they should be easily measured over a broad range of disease severities and in varied clinical settings. Ideally, the diagnostic test should assay for an essential component of proposed pathogenic mechanisms. Neutrophil collagenase and gelatinase are promising enzymes for diagnostic tests because of (1) their apparently central role in periodontal attachment loss and disease progression; (2) demonstrations of positive associations between enzyme levels and attachment loss and inflammation; and (3) availability of sensitive and specific assays to quantify these enzymes. However, much less data exist on reproducibility, diagnostic accuracy, and clinical use in longitudinal studies. In the future, more emphasis must be placed on the importance of appropriate study design for establishing the efficacy of collagenolytic enzymes as diagnostic tests.

Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites.

Intracrevicular plaque from periodontally-healthy individuals who had refrained from oral hygiene measures for 24 h prior to sampling, and subgingival plaque from diseased sites of patients with chronic periodontitis were screened by ELISA for the presence of Porphyromonas gingivalis and Treponema denticola. The samples were also subjected to the PerioScan test to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA). Of the 141 samples from periodontally-healthy sites, 73% contained T. denticola antigens and 78% P. gingivalis antigens, compared to 43% and 59%, respectively, in plaque samples from the 159 diseased sites. A positive reaction in the PerioScan test was obtained in 89% of plaque samples from diseased sites and in 60% of those from healthy sites. The correlation between the results of the two assays was poor in the case of intracrevicular plaque from healthy sites. However, with plaque samples from diseased sites, the results of the PerioScan test showed very strong correlation with those obtained with the ELISA, suggesting that the former may be a useful, rapid means of indicating the presence of T. denticola and P. gingivalis in such plaque samples.

Correlation between the BANA test and oral malodor parameters.

The purpose of the present investigation was to test the association between the BANA test (Perioscan, Oral-B), and oral malodor parameters. The subject population consisted of 52 Israeli adults, 43 of whom complained of oral malodor. Oral malodor measurements consisted of peak and steady-state volatile sulphide measurement by a portable sulphide monitor (Interscan Corp., model 1170), as well as organoleptic measurements of malodor from whole mouth, tongue, and saliva. Samples for the BANA test were obtained from four loci (shallow pocket, deep pocket, tongue dorsum, saliva); results were scored as negative (0), weak (1), or strong (2). BANA scores were significantly associated with odor-judge ratings, with the highest association obtained when BANA saliva scores and odor-judge saliva assessment were compared (r = 0.500; p < 0.001). BANA tests from the different loci were not significantly associated with sulphide monitor levels. Stepwise multiple-regression analysis of odor-judge measurements in terms of sulphide levels and average BANA scores showed that both log peak sulphide levels as well as BANA scores were significantly factored into the equations, yielding, in all cases, highly significant correlations (multiple r = 0.57, 0.50, and 0.59, respectively, with significance levels of 0.0001, 0.001, and < 0.0001, for whole mouth, tongue, and saliva malodor, respectively). The results suggest that the BANA scores are associated with a component of oral malodor which is independent of volatile sulphide measurements and suggest its use as an adjunct test to volatile sulphide measurement.

Prevalence of macroamylasaemia using polyethylene glycol precipitation as a screening method.

Four hundred and fifty-four subjects with hyperamylasaemia were screened for the presence of macroamylase using polyethylene glycol precipitation (PEG) and the Beckman automated amylase assay based on the hydrolysis of maltotetraose. Twenty-five subjects (5.5%) exhibited PEG precipitation values suggestive of macroamylasaemia (MA) (>52% loss of original amylase activity). Macroamylasaemia was confirmed by gel filtration chromatography (GFC) in 21/25 subjects, using albumin as a molecular weight marker. Biochemical and clinical details of the 21 subjects identified are presented and discussed. It is recommended that serum exhibiting more than 57% precipitation of the original amylase activity by PEG should be examined by GFC to confirm or exclude MA.

Diagnosis of periodontal diseases.

At the present time, the diagnosis and classification of periodontal diseases are almost entirely based on traditional clinical assessments. Supplemental quantitative and qualitative assessments of the gingival crevicular fluid and subgingival microflora can potentially provide useful information about the patient's periodontal disease. In certain situations, these supplemental risk-assessment tests may be particularly valuable in establishing the endpoint of therapy prior to placing patients on a periodontal maintenance program. Although the clinical utility of none of these tests has been validated, their further development is warranted. A genetic test for susceptibility to periodontitis has become commercially available. How best to use this and future host-based tests in clinical practice remains to be determined. Probing depth and clinical attachment loss measurements obtained with periodontal probes are practical and valid methods for assessing periodontal status. Computer-linked, controlled-force electronic periodontal probes are commercially available and are currently in use by some practitioners. Many of the logistical problems associated with subtraction radiography are being overcome and this powerful diagnostic tool may soon come into widespread use. Future developments in this and other imaging techniques are likely to have a profound effect on our approach to the diagnosis of periodontal diseases.