The requirement for gastrin measurements

In order to evaluate the clinical requirement for gastrin measurements, we examined all gastrin measurements requested over 1.5 years in a homogeneous population of 5.1 million inhabitants. Gastrin was quantitated with a radioimmunoassay that measured bioactive gastrins with equimolar potency. We received 1392 serum samples from 931 patients. In 394 samples from 121 patients the gastrin concentration was above the limit of the reference interval (50 pmol/l). Of the 121 patients, 19 were known Zollinger-Ellison patients followed for control of the therapy. In 11 previously unknown patients the gastrin analysis suggested presence of gastrin-producing tumours. Of these, four had classical Zollinger-Ellison syndromes, three had mixed endocrine tumours without peptic ulcer, and four were awaiting final confirmation of gastrinomas. Two vitiligo patients were hypergastrinaemic suggesting latent pernicious anaemia. Upon second measurement the plasma gastrin concentrations were within the reference interval in 14 previously hypergastrinaemic ulcer patients. In the remaining 75 patients the hypergastrinaemia was secondary to other gastrointestinal diseases. The results indicate that diagnosis, localization, and therapeutic control of gastrinomas require 200 gastrin measurements per million inhabitants per year. We suggest that this number be used in planning gastrin-assay services.     

ProBNP‐derived peptides in cardiac disease

The natriuretic peptides constitute a family of structurally related peptides that regulate fluid homeostasis, vascular tonus and growth. After the discovery of an endocrine component of the heart almost 25 years ago, the cardiac natriuretic peptides have now been fully accepted as useful markers in diverse aspects of cardiology including as diagnostic, therapeutic and prognostic markers of cardiac disease. In humans, atrial natriuretic peptide (ANP) and B‐type natriuretic peptide (BNP) are mainly synthesized and secreted by the failing heart, whereas the related C‐type natriuretic peptide (CNP) appears to be a local regulatory peptide secreted by the vascular endothelium. Accordingly, CNP is not a cardiac peptide. With the recent implementation of sensitive and specific immunoassays, increased plasma concentrations of proBNP‐derived peptides have now been associated with several cardiac conditions, where the major application today seems related to ventricular dysfunction. Recently, focus has also turned to ischemic heart disease, since myocardial hypoxia increases the local BNP gene expression. This review recapitulates the established clinical applications of measuring proBNP‐derived peptides in plasma. Furthermore, the evidence of increased cardiac BNP expression in ischemic heart disease will be emphasized. In turn, plasma measurement of proBNP‐derived peptides may still hold new possibilities in screening for coronary artery disease.     

Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay.

Recent studies on the gene sequence encoding the human pyloric antral hormone, gastrin, indicate a precursor of 101 residues. We have now raised antibodies to a synthetic analogue corresponding to (Tyr)-human progastrin COOH-terminal pentapeptide. The antibodies could be used in radioimmunoassay to measure this peptide, but they did not react with corresponding fragments of procholecystokinin, porcine progastrin, or other human progastrin-derived peptides, notably heptadecapeptide gastrin (G17), and 34-residue gastrin (G34). Radioimmunoassay of human antral and duodenal extracts revealed a major peak of activity that corresponded to the native COOH-terminal fragment of progastrin, and occurred in approximately equimolar amounts with COOH-terminal G17 immunoreactivity. In addition, there was a minor peak of apparently higher molecular weight material. In some gastrinomas the latter material was the predominant immunoreactive form, and it occurred in higher molar concentrations than any other form of gastrin. Digestion of this material with trypsin liberated peptides that reacted with antibodies specific for the NH2-terminus of G34, and G17. On this basis the high molecular weight component was identified as a form of gastrin that extended from the COOH-terminus of the precursor to a point at least beyond the NH2-terminus of G34, and probably included the entire progastrin sequence. The results suggest differences in posttranslational processing pathways of progastrin in antrum, duodenum, and gastrinomas. They also indicate that the present experimental approach allows the identification of progastrin-like substances, which should open the way to studying the mechanisms of gastrin biosynthesis.     

ProBNP-derived peptides in cardiac disease

The natriuretic peptides constitute a family of structurally related peptides that regulate fluid homeostasis, vascular tonus and growth. After the discovery of an endocrine component of the heart almost 25 years ago, the cardiac natriuretic peptides have now been fully accepted as useful markers in diverse aspects of cardiology including as diagnostic, therapeutic and prognostic markers of cardiac disease. In humans, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are mainly synthesized and secreted by the failing heart, whereas the related C-type natriuretic peptide (CNP) appears to be a local regulatory peptide secreted by the vascular endothelium. Accordingly, CNP is not a cardiac peptide. With the recent implementation of sensitive and specific immunoassays, increased plasma concentrations of proBNP-derived peptides have now been associated with several cardiac conditions, where the major application today seems related to ventricular dysfunction. Recently, focus has also turned to ischemic heart disease, since myocardial hypoxia increases the local BNP gene expression. This review recapitulates the established clinical applications of measuring proBNP-derived peptides in plasma. Furthermore, the evidence of increased cardiac BNP expression in ischemic heart disease will be emphasized. In turn, plasma measurement of proBNP-derived peptides may still hold new possibilities in screening for coronary artery disease.     

Heart failure and neuroendocrine activation: diagnostic,prognostic and therapeutic perspectives

The important neuroendocrine systems involved in heart failure are reviewed with special emphasis on their possible role in pathophysiology and their relation to prognostic and diagnostic information. Plasma levels of noradrenaline (NA), renin, vasopressin, endothelin‐1, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and tumour necrosis factor‐α (TNF‐α) are all elevated in heart failure. Activity of the sympathetic nervous system as reflected by NA is correlated to mortality and seems to possess independent prognostic information. Several studies have now documented the beneficial effect of β‐blockade in chronic heart failure (CHF). Renin seems to be a poor prognostic marker in CHF possibly because of the interference with diuretic treatment, angiotensin converting enzyme (ACE)‐inhibitors and angiotensin II antagonist, and probably also because of the significance of tissue renin‐angiotensin system (RAS), poorly reflected by plasma renin. On the other hand, several large‐scale trials with ACE‐inhibitors and angiotensin II antagonists have demonstrated reduced mortality and morbidity in CHF. Plasma vasopressin does not seem to possess prognostic information but testing of non‐peptide antagonists is ongoing. Endothelin‐1 seems to have independent prognostic information and endothelin receptor antagonists may represent a therapeutic possibility. The natriuretic peptides ANP and BNP are correlated to prognosis and possess independent information. Brain natriuretic peptide and N‐terminal ANP seem to increase early, i.e. in asymptomatic heart failure. Plasma BNP being more stable than ANP is therefore a promising measure of left ventricular dysfunction. Increase in ANP and BNP, potentially beneficial, may be achieved by administration of neutral endopeptidase inhibitors, at present an unsettled therapeutic possibility. Several cytokines are increased in heart failure and especially TNF‐α has drawn attention. Experimental studies suggest that TNF‐α is important in the pathophysiology of heart failure and preliminary studies indicate that inhibition of TNF‐α seems to be a possible therapeutic approach. Thus, neuroendocrine markers seem to (i) have a role in diagnosis and classification of heart failure, (ii) be useful in providing a ‘neuroendocrine profile’ which enlightens different aspects of heart failure, and therefore (iii) in the future probably will be valuable in the choice of medical treatment of the individual patient. In addition to β‐blockers, ACE‐inhibitors and angiotensin II antagonists several new drugs based on neuroendocrine modification are on their way and might become important in the future.     

PreproVIP-derived peptides in tissue and plasma from patients with VIP-producing tumours

To elucidate the biosynthetic processing of the precursor for vasoactive intestinal peptide (prepro-VIP) in tumours producing VIP we have used newly developed radioimmunoassays directed against the five functional domains of the VIP precursor molecule: preproVIP 22-79, peptide histidine methionine (PHM), preproVIP 111-122, VIP and preproVIP 156-170 in combination with HPLC to identify and quantify the peptides in tumour specimen and plasma from patients with the watery diarrhoea syndrome. Elevated quantities of all the five peptides were found in the 13 tumours (nine neurogenic tumours, one pheochromocytoma, three pancreatic carcinomas) examined. The preproVIP derived peptides were expressed in non-equimolar amounts and the relative proportion of the various peptides differed markedly from tumour to tumour. The pheochromocytoma was the only tumour type which contained large amounts of preproVIP 156-170 in comparison with the other peptides. A proportion of the VIP precursor which varied from 7% to 73% followed a pathway in which the dibasic conversion site after PHM was uncleaved as evidenced by the presence of PHV, a C-terminally extended form of PHM. It was also found that unlike normal tissue a fraction of the C-terminal VIP precursor peptide, preproVIP 156-170, was having its C-terminal lysine residue removed during processing. The findings indicate that various post-translational processing pathways of preproVIP exist. All the peptide sequences produced in the tumour tissue were secreted as evidenced by their presence in plasma in elevated concentrations. The plasma levels of preproVIP 22-79, preproVIP 111-122 and PHV exceeded those of the remaining preproVIP-derived peptides suggesting that determination of these peptides in patients with VIP-secreting tumours may be better markers than VIP.     

Immunochemical studies on gastrins in the urine.

The concentration and molecular form of gastrin in urine were determined radioimmunochemically. Urine from hypergastrinaemic patients (Zollinger-Ellison syndrome and pernicious anaemia) contained gastrins corresponding to the serum components I and II. The excretion of gastrin increased with increasing gastrin concentrations in serum. Urine from six subjects with normal concentrations of gastrin in serum contained "apparent" gastrin immunoreactivity which could not be removed by specific immunoabsorption. No gastrin was detectable by gel filtration of desalted and concentrated urine from normal subjects. The apparent immunoreactivity was due partly to interference by sodium chloride. The results indicate that hypergastrinaemic patients, in contrast to normal subjects, excrete gastrins in the urine.     

The effect of the mammalian neuropeptide, gastrin-releasing peptide (GRP), on gastrointestinal and pancreatic hormone secretion in man

Gastrin-releasing peptide, a newly isolated mammalian peptide similar in its structure and actions to the amphibian peptide, bombesin, has recently been localized to nerves in the brain, gut and pancreas. The present study investigates its effects on gut and pancreatic peptides in man. Intravenous infusion of 0.7 and 2.9 pmol min-1 kg-1 produced significant elevation of plasma gastrin, cholecystokinin-like immunoreactivity and neurotensin. It was found also to potentiate glucose-dependent insulin secretion. Its specific location in nerve fibres in the proximal gut and pancreas and its selective effect on gastroenteropancreatic peptides may favour its role as a physiological regulatory neuropeptide.     

Gastrinoma – morphological aspects

Gastrinomas are defined as gastrin producing tumors that are associated with an elevated fasting gastrin serum level, a positive gastrin secretin stimulation test and certain clinical symptoms, e.g. recurrent peptic ulcer disease and occasionally diarrhea, the so-called Zollinger-Ellison syndrome. Most gastrinomas occur in the duodenum (approx. 70%) and not in the pancreas. The duodenal gastrinomas are small, and when they occur in association with the genetic syndrome of multiple endocrine neoplasia type 1 (MEN1), they are multicentric and originate from precursor lesions. The prognosis of duodenal gastrinomas is better than that of pancreatic gastrinomas, since despite early lymph node metastasis they progress slowly to liver metastasis.     

Neuropeptides: Metabolism to Bioactive Fragments and the Pharmacology of Their Receptors

The proteolytic processing of neuropeptides has an important regulatory function and the peptide fragments resulting from the enzymatic degradation often exert essential physiological roles. The proteolytic processing generates, not only biologically inactive fragments, but also bioactive fragments that modulate or even counteract the response of their parent peptides. Frequently, these peptide fragments interact with receptors that are not recognized by the parent peptides. This review discusses tachykinins, opioid peptides, angiotensins, bradykinins, and neuropeptide Y that are present in the central nervous system and their processing to bioactive degradation products. These well‐known neuropeptide systems have been selected since they provide illustrative examples that proteolytic degradation of parent peptides can lead to bioactive metabolites with different biological activities as compared to their parent peptides. For example, substance P, dynorphin A, angiotensin I and II, bradykinin, and neuropeptide Y are all degraded to bioactive fragments with pharmacological profiles that differ considerably from those of the parent peptides. The review discusses a selection of the large number of drug‐like molecules that act as agonists or antagonists at receptors of neuropeptides. It focuses in particular on the efforts to identify selective drug‐like agonists and antagonists mimicking the effects of the endogenous peptide fragments formed. As exemplified in this review, many common neuropeptides are degraded to a variety of smaller fragments but many of the fragments generated have not yet been examined in detail with regard to their potential biological activities. Since these bioactive fragments contain a small number of amino acid residues, they provide an ideal starting point for the development of drug‐like substances with ability to mimic the effects of the degradation products. Thus, these substances could provide a rich source of new pharmaceuticals. However, as discussed herein relatively few examples have so far been disclosed of successful attempts to create bioavailable, drug‐like agonists or antagonists, starting from the structure of endogenous peptide fragments and applying procedures relying on stepwise manipulations and simplifications of the peptide structures.     

Supersensitive gastrin assay using antibodies raised against a cholecystokinin homolog

Peptide hormones may occur in particularly low amounts in samples from small animals. Hence, in a rat microdialysis study conventional immunoassays were not sufficiently sensitive to measure gastrin in the dialysis samples. We therefore exploited the observation that antibodies raised against the homologous hormone cholecystokinin (CCK) occasionally bind gastrin peptides with significantly higher affinity than the proper ligand. The immunoassay thus established could detect 1.0 pmol/l in 15 μl microdialysate, which corresponds to 23 attomol gastrin. Such detection limit is five-fold lower than that obtained with the most avid conventional gastrin antibodies. The results may encourage similar approaches for other peptides using homologue-raised antibodies when supersensitivity is required.     

Measurement of nonsulfated cholecystokinins

Abstract

Most proteins undergo posttranslational modifications that govern the function of the protein. In synchrony, correspondingly unmodified proteins that are functionally silent or act differently may also be synthesized. The gut hormone precursor, procholecystokinin (proCCK) is an example of a protein that is heavily modified. An essential modification is O-sulfation of Y₇₇, which is necessary for the gallbladder emptying effect of CCK peptides via the CCK_A-receptor. In order to examine possible in vivo synthesis also of nonsulfated CCK, we have established a two-step analysis that requires tryptic cleavage at a defined processing site in proCCK (R₇₅-D₇₆) followed by monospecific RIA-measurement of the then exposed nonsulfated N-terminal sequence of CCK-8 (DYMGW…). The analysis shows that endocrine cells in the gut synthesize nonsulfated CCK peptides (?58, ?33, ?22, and ?8) in the order of 20–35% of the corresponding sulfated CCKs. Since nonsulfated CCK peptides are full agonists of the CCK_B-receptor, the assay has revealed a hitherto unrecognized gut hormonal peptide system. The assay may prove useful in the diagnosis and control of diseases with hyperCCKemia. This includes CCK-producing neuroendocrine tumors such as the recently described CCKomas and medullary thyroid C-cell carcinomas.     

Biochemistry of pro-B-type natriuretic peptide-derived peptides: the endocrine heart revisited

BACKGROUND: Since the discovery of cardiac hormones almost 25 years ago, a vast amount of clinical research has identified the cardiac natriuretic peptides and their precursors as markers of heart failure. It even seems likely that the pro-B-type natriuretic peptide (proBNP)-derived peptides in plasma may become the most frequently measured peptides in the daily diagnosis and control of therapy. In contrast, the biochemistry of the peptides has received less attention. METHODS: Published data available on the National Library of Medicine (NLM) were used as the basis for the review. OUTCOME: This review shows that the present understanding of the biochemistry of peptides is far from complete. In particular, cellular synthesis, including posttranslational precursor maturation, is poorly understood. Moreover, elimination of the precursor fragments is unknown. Elucidation of the molecular heterogeneity of proBNP products will therefore contribute to the understanding of the endocrine heart and may also have important diagnostic consequences. Above all, the different proBNP-derived peptides may not always be equal markers of the same pathophysiologic processes. A different metabolism and peripheral elimination may also impose new and peptide-specific limitations for diagnostic use. CONCLUSIONS: It is necessary to focus more on the biology of the proBNP-derived peptides. In turn, new insight into the biochemistry could pave the way for more sensitive and disease-specific assays in the clinical setting.     

Human immunodeficiency virus associated thrombotic thrombocytopenic purpura,a clinical conundrum

HIV complicates the diagnostic and therapeutic approaches to idiopathic thrombotic thrombocytopenic purpura (TTP), prompting debate in the literature regarding the benefit of plasma exchange versus simple plasma infusion. Herein we present a case of HIV‐TTP, initially treated conservatively with plasma infusion but because of progressive neurologic decline, required urgent plasma exchange for resolution of hematologic derangements and neurologic sequelae. Based on the available literature, there appears to be a spectrum of HIV‐associated TTP disorders. Patients with advanced HIV disease and opportunistic infections who present with thrombotic microangiopathy tend to respond to simple plasma infusion, while patients with less progressive HIV disease tend to behave like those with idiopathic TTP, requiring plasma exchange rather than simple plasma infusion. This article illustrates that in patients with HIV‐TTP who do not respond to plasma infusion, early escalation to plasma exchange may help avoid life‐threatening complications such as seizures and even death.     

Differential responses of plasma atrial and brain natriuretic peptides to acute alteration in atrial pressure in pigs

To describe the differential dynamic responses of atrial natriuretic peptide (ANP), N-terminal proatrial natriuretic peptide (Nt-proANP) and brain natriuretic peptide (BNP) to acute changes in atrial pressure, 6 pigs were studied during and after a 24-h period of rapid atrial pacing (225 bpm). During pacing, left atrial pressure increased acutely. ANP plasma concentrations showed a sharp initial peak followed by a decline, but remained significantly increased throughout the 24-h period. Nt-proANP followed a smoother pattern, increasing significantly only after 24 h. BNP increased significantly after 8 h after pacing and even more after 24 h. An opposite but similarly differential pattern of peptide responses was found in the post-pacing period. The different responses in ANP, Nt-proANP and BNP plasma concentrations may reflect the different mechanisms of regulation of secretion as well as plasma clearance. If the present findings reflect the acute clinical situation in humans, they may be of diagnostic relevance. An isolated ANP elevation would indicate a recent acute pressure increase, while elevation of two or more natriuretic peptides would point to a pressure increase of longer duration.     

Osteocalcin: diagnostic methods and clinical applications

Osteocalcin is a small (Mr 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin.     

Elevated procalcitonin and C‐reactive protein as potential biomarkers of sepsis in a subpopulation of thrombotic microangiopathy patients

Thrombotic microangiopathy (TMA) comprises a group of microvascular thrombosis syndromes associated with multiple pathogenic factors. Deficient activity of ADAMTS13 is a pathogenic factor in a subset of TMA patients that provides a strong rationale for plasma exchange treatment. However, the subset of TMA patients with normal ADAMTS13 activity remains a heterogeneous group of patients in which the appropriate treatment is not well understood. In addition to the common forms of TMA thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome, the differential diagnosis of TMA may include sepsis, autoimmune disorders, and disseminated intravascular coagulation. Optimal treatment of TMA depends on timely recognition of treatable pathogenic factors. We hypothesized that sepsis is a rapidly identifiable pathogenic factor in a subset of TMA patients. To test this hypothesis, we retrospectively measured the rapid biomarkers of sepsis C‐reactive protein (CRP) and procalcitonin (PCT), in a repository of pretreatment plasma samples from 61 TMA patients treated with plasma exchange. Levels were analyzed in 31 severely ADAMTS13‐deficient and 30 ADAMTS13‐normal patients. None of the 31 patients with severe deficiency of ADAMTS13 had elevated PCT. However, 11 of 30 (37%) non‐ADAMTS13‐deficient patient samples were strongly positive for PCT. These patient samples also had a >10‐fold higher median CRP level than patients with normal PCT. We conclude that rapid assays may help identify sepsis in a subset of TMA patients. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Diagnosis of heart failure using urinary natriuretic peptides

In the present study, we assessed the use of urinary natriuretic peptides [N-terminal proatrial natriuretic peptide (N-ANP) and N-terminal pro-brain natriuretic peptide (N-BNP) and C-type natriuretic peptide (CNP)] in the diagnosis of heart failure. Thirty-four consecutive hospitalized heart failure patients (median age, 75.5 years; 14 female) were compared with 82 age- and gender-matched echocardiographically normal controls. All subjects provided plasma and urine specimens. Plasma was assayed for N-BNP, and urine was assayed for N-ANP, N-BNP and CNP. The diagnostic efficiency of peptides was assessed using receiver operating characteristic (ROC) curve analysis. All three urinary natriuretic peptides were significantly elevated in heart failure patients ( P <0.001). Urine N-BNP was correlated with plasma N-BNP ( r (s)=0.53, P <0.0005). Areas under the ROC curves for urinary N-ANP, N-BNP and CNP were 0.86, 0.93 and 0.70 and for plasma N-BNP was 0.96. Correcting urinary peptide levels using urine creatinine produced ROC areas of 0.89, 0.93 and 0.76 respectively. A urine N-BNP level cut-off point of 11.6 fmol/ml had a sensitivity and specificity for heart failure detection of 97% and 78% respectively, with positive and negative predictive values of 64.7 and 98%. In conclusion, although all three natriuretic peptides were elevated in urine in heart failure, urinary N-BNP had diagnostic accuracy comparable with plasma N-BNP. Use of urinary N-BNP for heart failure diagnosis may be suitable for high-throughput screening, especially in subjects reluctant to provide blood samples.     

Structure of the sulfakinin cDNA and gene expression from the Mediterranean field cricket Gryllus bimaculatus

The sulfakinins are multifunctional insect neuropeptides displaying sequence similarities with the gastrin/ cholecystokinin (CCK) peptide family. In vertebrates, the peptides gastrin and CCK are involved in the regulation of digestion and food-intake. In this study sulfakinin cDNA was cloned and sequenced from the Mediterranean field cricket Gryllus bimaculatus. The cDNA encodes two peptides flanked by endoproteolytic processing sites, designated GrybiSKI (QSDDYGHMRFG) and GrybiSKII (EPFDDYGHMRFG). The peptides include the characteristic amino acid Tyr, which is potentially sulphated, and a Gly, as a recognition site for amidation yeilding the common C-terminal amino acid sequence of the sulfakinin peptide family. RT-PCR studies indicate an expression of the gene restricted to the brain, with a constant level of expression throughout the last larval stage, but showing an age-dependent decrease of expression in adult females.     

Peptide‐mediated targeted drug delivery

Targeted drug delivery to specific group of cells offers an attractive strategy to minimize the undesirable side effects and achieve the therapeutic effect with a lower dose. Both linear and cyclic peptides have been explored as trafficking moiety due to ease of synthesis, structural simplicity, and low probability of undesirable immunogenicity. Peptides derived from sequence of cell surface proteins, such as intercellular adhesion molecule‐1 (ICAM‐1), LHRH, Bombesin, and LFA‐1, have shown potent binding affinity to the target cell surface receptors. Moreover, peptides derived from ICAM‐1 receptor can be internalized by the leukemic T‐cells along with the conjugated moiety offering the promise to selectively treat cancers and autoimmune diseases. Systematic analyses have revealed that physicochemical properties of the drug–peptide conjugates and their mechanism of receptor‐mediated cellular internalization are important controlling factors for developing a successful targeting system. This review is focused on understanding the factors involved in the development of an effective drug–peptide conjugate with an emphasis on the chemistry and biology of the conjugates. Reported results on several promising drug–peptide conjugates have been critically evaluated. The approaches and results presented here will serve as a guide to systematically approach targeted delivery of cytotoxic drug molecules using peptides for treatment of several diseases. © 2010 Wiley Periodicals, Inc. Med Res Rev, 32, No. 3, 637‐658, 2012