The correct diagnosis and clear understanding of disease pathogenesis are the key factors for success of pharmacotherapy. Clinical biochemistry is therefore regarded as one of the basic pillars of medical science that enables the clinicians to perceive the underlying mechanisms of various disease conditions. Biomarkers are invaluable tools available to clinicians and biochemists for diagnosis/prognosis of diseases as well as for the evaluation of treatment planning. Biomarkers also play an important role in basic and clinical research to reveal new insights in various disciplines of medical sciences. The significance and applications of our research findings are categorically summarized below:
Patients with type-2 diabetes often exhibit an atherogenic lipid profile (high triglycerides and low HDL cholesterol). Diabetic patients with accompanied dyslipidemia are therefore soft targets of cardiovascular deaths. An early intervention to normalize circulating lipids has been shown to reduce cardiovascular complications and mortality. Most of the diabetic patients carry unnoticed dyslipidemia characterized by increased levels of triglycerides (TG) and LDL and decreased HDL. Glycated hemoglobin (HbA1c) is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding 2-3 months. We have examined the relationship between glycemic control and serum lipid profile and evaluated the relevance of HbA1c as an indicator of circulating lipids in type-2 diabetic patients. Our results suggest that dual biomarker capacity of HbA1c (glycemic control as well as lipid profile indicator) can be utilized for screening high risk diabetic patients for timely intervention with lipid lowering drugs. Furthermore, there were no significant interactions between sex or age and HbA1c with respect to lipid profile suggesting the validity of HbA1c for predicting dyslipidemia irrespective of patient’s gender and age. These results clearly suggest that HbA1c endures the ability of predicting serum lipid profile in both male and female diabetic patients.
Gestational diabetes mellitus (GDM) is a common medical complication during pregnancy and is associated with adverse fetal and maternal outcomes that can be prevented by timely diagnosis and management. Routine screening of GDM is an important aspect of antenatal care in order to minimize its serious consequences. Oral glucose tolerance test (OGTT) is the gold standard for the diagnosis of GDM however the routine application of OGTT for screening of GDM is hampered by its high cost, lengthy procedure, growing number of patients and patients’ noncompliance. Simple approaches are therefore sought to minimize the use of OGTT without compromising the likelihood of diagnosing GDM. We have observed huge fluctuations in fasting blood glucose (FBG) and to a lesser extent in serum fructosamine which clearly indicate that both these tests individually cannot be regarded as safe screening markers for GDM on the basis of particular cut-off values. Interestingly the fluctuations in FBG were found to be independent of fluctuations in serum fructosamine. Thus, the paired values of FBG and fructosamine would be more advantageous than their individual values in filtering high-risk patients on whom OGTT should be performed for the confirmation of GDM. The results of our clinical studies have shown that the use of paired values of fructosamine and fasting or random blood glucose offer a sensitive and safe strategy for short listing the high risk patients and therefore avoiding unnecessary OGTT in a large number of cases.
Sepsis and its sequel are often lethal and considered to be the leading causes of mortality in intensive care units. The cascade of events initiating from infection to septic shock and multi-organ failure is poorly understood. Phagocytosis is the first line of host defense against foreign antigens including bacteria. Polymorphonuclear leukocytes (PMNs) such as neutrophils and macrophages are responsible for inactivating and clearing bacteria and microbial-related noxious products using the process of phagocytosis. The recruitment and activation of PMNs at the site of infection are essential for effective host defense. However, prolonged activation of PMNs results in excessive generation of pro-inflammatory cytokines and reactive oxygen species (ROS) that can lead to progressive damage of host tissue. Although sepsis-induced infiltration and activation of PMNs are well documented concurrent profiles of circulating and extravasated PMNs are not well studies. We have clearly demonstrated sepsis-induced alterations in both blood and peritoneal fluid PMNs and their quantitative assessment might help in disease evaluation and designing effective therapies.
Fluconazole is a well-tolerated anti-fungal drug with demonstrated ability to reversibly penetrate into human PMNs. Fluconazole has been shown to improve survival and reduce multi-organ damage in experimental and clinical septic shock. Since fluconazole has no inherent anti-bacterial properties, its beneficial effects in bacteremia have been attributed to its action on the modulation of neutrophils sequestration and activation. However, the findings of earlier studies regarding the effect of fluconazole on the phagocytic response of PMNs are controversial. Thus to better understand the nature of interaction between fluconazole and PMNs in sepsis, we evaluated the effect of fluconazole on phagocytic activity of PMNs in a rat model of septic shock. The findings of this study showed that fluconazole protected rats against septic shock by inhibiting PMN-mediated inflammatory cascade without compromising their phagocytic activity. In another study we observed a significant increase in myeloperoxidase (MPO) activity in lungs and peritoneal fluid of rats with septic shock indicating massive infiltration and activation of neutrophils in these sites. All these parameters were significantly and dose-dependently attenuated by fluconazole treatment suggesting that drugs with the ability to down-regulate inflammatory cascade can have beneficial effects in septic shock.
Psoriasis vulgaris is a chronic inflammatory skin disease of unknown etiology. Currently, one of the main areas of research in psoriasis field concerns the role of cytokines in the pathogenesis of this disease. Although cytokine-mediated response is an essential part of the natural protective mechanism, excessive production of pro-inflammatory cytokines or cytokines in the wrong biological context are associated with the pathology in a wide range of diseases including psoriasis. The findings of our clinical studies suggest that an array of cytokines may be considered as useful follow-up marker for monitoring of psoriasis patients and optimizing therapeutic strategies. We have also noticed significant association between HLA-C and susceptibly to psoriasis vulgaris. To our knowledge, no such attempt had been made earlier to investigate HLA associations with psoriasis in Saudi population despite a high prevalence of this disease among Saudis.
Dyskinesia is a potentially irreversible movement disorder associated with long-term administration of neuroleptic drugs. Iminodipropionitrile (IDPN) is a neurotoxic compound that produces motor syndrome in rodents, which resembles human idiopathic dyskinesia and has been used as an experimental model for drug-induced dyskinesia. IDPN-induced dyskinesia is also termed as ECC (excitation, chorea, circling) syndrome and characterized by repetitive head movements, retropulsion, circling, back walking, hyperactivity and swimming deficits. Because of the unique array of neurobehavioral abnormalities, IDPN is considered as one of the most suitable compounds for validation of functional observational battery (FOB) and motor activity for screening of neurotoxic drugs. The mechanism of IDPN-induced dyskinesia appears to be complex and multifactorial. We therefore performed extensive research to understand the potential mechanisms involved in the pathogenesis of IDPN-induced neurotoxicity. Our results showed that IDPN-induced motor deficits are associated with increased oxidative stress, alteration in neurotransmitters and vestibular hair cell degeneration.
Parkinson’s disease (PD) is a commonly occurring neurodegenerative disorder that produces muscular rigidity, bradykinesia, tremor of resting limbs and loss of postural balance. The basic neuropathology of PD involves degeneration of pigmented neurons in substantia nigra resulting in depletion of dopamine (DA). The agents with the ability to interfere with the synthesis, release, uptake, metabolism or drug-receptor interaction of DA have been shown to alter the course of experimental Parkinsonism. Peripheral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) in C57 black mouse and intrastriatal injection of 6-hydroxydopamine (OHDA) are the most extensively used animal models of PD. Important roles of reactive oxygen species (ROS), inflammatory mediators and impaired immune system have been implicated in PD. A significantly less phagocytic function of the PMNs has earlier been observed in patients with PD.
We have evaluated the time-course effects of neurotoxin MPTP in a mouse model of Parkinson’s disease suggesting that an early stage pro-inflammatory response following MPTP might trigger a chain of potentially toxic pathways mediated by ROS leading to progressive neuronal damage. Our results also showed significant reversal of MPTP- and OHDA-induced dopaminergic toxicity by PLA2 inhibitor quinacrine implicating an important role of PLA2 in experimental Parkinsonism. On the other hand, dipyridamole (an adenosine transport inhibitor) was found to enhance MPTP-induced neurotoxicity suggesting a caution in administering dipyridamole or other adenosine analogs in patients suffering from Parkinson’s disease. Magnesium also significantly and dose-dependently exacerbated MPTP-induced motor dysfunction, lethality and depletion of striatal DA suggesting the ability of magnesium to modify behavioral and biochemical alterations in experimental Parkinsonism.
Huntington’s disease (HD) is a chronic progressive autosomal dominant neurodegenerative disorder that is characterized by striatal-specific degeneration. In spite of extensive research this devastating hereditary disease remains incurable, warranting further studies to determine the cause and cure of HD. It has been reported that nicotinic acetylcholine receptor (nAchR) in the central nervous system (CNS) is a new potential therapeutic target for the management of neurodegenerative diseases. We have demonstrated the protective effects of nAchR agonist, nicotine, against 3-nitropropionic acid (3-NP) induced biochemical, behavioral and histological changes in a rat model of Huntington’s disease. Our findings further new insights of the mechanisms of HD that might help to design novel therapies for this disabling neurodegenerative disease.
The series of biochemical, pathological, immunological events that occur in the period of hours to days following initial traumatic injury might be more hazardous than the damage caused by the primary impact. This secondary phase consists of loss of autoregulation, elevated intracranial pressure, systemic hypotension, hemorrhage, thrombosis, edema, inflammation and vasospasm leading to hypoperfusion and ischemia. Adenosine is a natural protector and its agonists alleviate ischemic brain injury. Caffeine is a broad-spectrum adenosine receptor antagonist and exerts its pharmacological effects by blocking adenosine A1 and A2 receptors. The potential of caffeine to improve alertness and counteract alcohol-related driving impairment and accidents has been demonstrated earlier. We have evaluated the effects of caffeine on neurological outcome following experimental head injury in rats. We have shown that caffeine significantly and dose-dependently increases neutrophil infiltration, edema, disruption of blood-brain-barrier, and lipid peroxidation in traumatic brain tissue. There was severe hemorrhage and neuronal degeneration in the injured brain hemisphere of caffeine-treated rats as compared to rats in injury-alone group.
In contrast to high mortality associated with head injury, patients with spinal cord injury often suffer partial disability or complete paralysis with many physical, psychological and social ramifications. We have evaluated the effects of aluminum on biochemical, neurobehavioral and electrophysiological changes in a rat model of spinal cord injury. The daily dietary intake of aluminum in humans from food and water ranges between 3 and 100 mg, and a serum aluminum level up to 10 mg/l is considered safe. Ingestion of aluminum containing antacids would cause an additional exposure of 5 g of aluminum per day which might lead to a 10-fold increase in serum aluminum in an individual with normal kidney function. Whereas the use of antacids and phosphate-binders in patients with impaired renal function results in a 40- to 100-fold increase in these serum levels. The increase in aluminum body burden may lead to neurotoxicity, nephrotoxicity, osteodystrophy and hyprochromic anemia. We have evaluated the effects of aluminum on biochemical, neurobehavioral and electrophysiological changes in a rat model of spinal cord injury. The recovery phase was significantly and dose-dependently retarded by aluminum. Aluminum also exacerbated the depletion of vitamin E in spinal cord of injured rats suggesting its ability to aggravate oxidative stress mediated injury following neurotrauma.
Snake Venom Toxicity
Echis pyramidum (EP) is a poisonous snake whose bite causes massive edema, pain, hemorrhage and necrosis in the victims. The lethal effects of snake venom are largely attributed to its active ingredient PLA2. Phospholipids hydrolysis by PLA2 enzyme releases arachidonic acid whose metabolism results in the formation of potentially toxic reactive oxygen species and lipid peroxides. To better understand the pathogenesis of snake envenomation we studied the effect of EP venom on time-course of lipid peroxidation in different vital organs (brain, heart, kidney, liver and lung) of mice. The results showed that a single injection of EP caused significant lipid peroxidation in all the organs studied. In all the organs, the onset of lipid peroxidation was as early as 1 hour and persisted for several hours suggesting an important role of oxidative stress in the cytotoxicity of EP venom.
Recently statins appear to have additional benefits beyond their lipid lowering effects which has led to the interest in using this class of drugs outside the realm of cardiovascular disease. We have shown gastroprotective effects of simvastatin (SIM) in rats. The antiulcer activity of SIM was associated with significant attenuation of adverse effects of ethanol on gastric wall mucus, glutathione (GSH) and myeloperoxidase (MPO). SIM modified the gastric nitric oxide (NO) levels and reversed ethanol-induced decrease in glutathione-S-transferase and increase in superoxide dismutase and catalase activities. We have also made a comparative evaluation of mast cell stabilizers nedocromil sodium (NDS) and sodium cromoglycate (SCG) in experimental gastric and duodenal ulcers in rats. The cytoprotective effects of these two drugs were accompanied by the attenuation of ethanol-induced depletion of gastric wall mucus and GSH. The differential effects of NDS and SCG against various gastric lesions rationalize the possible benefits of a combined therapy for the treatment of complex gastroduodenal ulcers.
Bromophenacyl bromide (BPB), a PLA2 inhibitor was also studied for its ability to inhibit gastric secretion and to protect chemically induced gastric and duodenal ulcers in rats. The results of this study demonstrate that BPB produces a dose-dependent inhibition of gastric acid secretion, acidity and gastric mucosa in rats. BPB also protected against cysteamine-induced duodenal ulcers. The antiulcer activity of BPB was associated with significant inhibition of ethanol-induced depletion of gastric wall mucus, GSH and MPO. These findings point towards the mediation of sulfhydryls in BPB-induced gastrointestinal cytoprotection.
We have also developed a computer method for quantitation of gastric lesions. The technique of direct scanning of stomach samples sandwiched in a transparent plastic folder has been introduced for simple, rapid, cost-effective and efficient morphological evaluation of gastric lesions. This method has been adopted by other scientists to evaluate the effect of drugs on gastric lesions.
Renal Transplant / Nephrotoxicity
Calcitonin is a 32 amino acid peptide hormone that is synthesized and secreted by the C-cells of thyroid gland. High levels of serum calcitonin have earlier been noticed in severe infections and inflammations suggesting serum calcitonin as a useful marker for these conditions. Although a number of calcitonin-based studies have been undertaken in renal failure and hemodialysis the role of calcitonin in renal transplant remains unclear. We therefore determined serum calcitonin in 60 renal transplant patients and the same number of age-matched controls. We also measured alkaline phosphatase, calcium, phosphate, albumin, urea, and creatinine in these subjects. Our results showed that the level of serum calcitonin is markedly elevated in renal transplant patients, this elevation being more significant in female patients. Among the other parameters, only urea and creatinine were significantly higher in transplant patients as compared to controls.
Cyclosporin (CsA) is a commonly used immunosuppressant in renal transplantation. Nephrotoxicity is one of the most significant side-effects associated with CsA therapy and its prolonged usage may lead to irreversible renal damage. Thus novel protective agents to counteract CsA nephrotoxicity without altering its immunosuppressive effect are badly needed to ensure the safe use of CsA. We have studied the effects of antioxidant drugs, N-acetylcysteine (NAC) and quinacrine (QNC) on CsA-induced nephrotoxicity in rats. Our results showed that both NAC and QNC significantly protected rats against CsA-induced increase in blood urea nitrogen (BUN) and serum creatinine. These drugs also significantly reversed the effects of CsA on free radical biomarkers suggesting the role of oxidative stress in CsA-induced nephrotoxicity and beneficial effect of antioxidants. In another study we observed synergistic effects of pregnancy and CsA on activation of PMNs resulting in massive and sustained generation of ROS that could be deleterious to both host and fetus. Thus, a combination of pregnancy, CsA therapy and infection or allergic condition should be considered risky and these patients should be closely monitored.
Environmental and occupational exposure of benzene has been associated with serious health hazards. Although part of benzene is excreted intact from the body, a major portion of benzene is converted into several metabolites including S-phenyl mercapturic acid (S-PMA), trans, trans-muconic acid (t,t-MA), phenol, catechol and hydroquinone. Since the relative production of these metabolites is directly proportional to benzene level, they are used a sensitive biomarkers of benzene exposure. Estimation of un-metabolized benzene in urine has also been suggested as a reliable indicator of its exposure. Chromatographic methods are efficiently used for the analysis of benzene and its metabolites. Solid-phase microextraction (SPME) presents extraordinary advantages over traditional extraction methods because it integrates sampling, extraction, pre-concentration, and introduction of sample in a single stage, which significantly minimizes the use of solvents. Moreover, the requirement of small amount of sample for SPME renders the technique highly suitable for the analysis of benzene in biological fluids. Gas chromatography (GC) appears to be a method of choice for the analysis of benzene whereas high-performance liquid chromatography (HPLC) is commonly applied for the estimation of benzene metabolites to assess benzene exposure in humans. We have reviewed the human and animal toxicities of benzene as well as chromatographic methods for the determination of benzene and its metabolites.
Bioassay Method Development
Attainment of high sensitivity is of prime concern particularly when assessing the biomarkers in tiny samples (e.g. 3-5 mg) such as tissue biopsies or discrete regions of rodent brain. Tyrosine hydroxylase (TH) is the primary enzyme involved in the biosynthesis of catecholamine neurotransmitters in the brain. TH has been recognized as a sensitive and reliable biomarker of neurodegeneration. We have demonstrated the crucial roles of assay buffer pH and cofactor concentration on the sensitivity of radiochemical assay of TH in rodent brain. An optimal pH range was found to be 5.5-6.0 whereas TH activity was significantly inhibited at pH 5.0 and pH 6.8. A significantly high TH activity was observed with 1.5 mM 6-MPH4, whereas higher concentrations of cofactor (3.0-4.5 mM) inhibited TH activity. The TH assay was validated for striatal samples from rats and mice brains.
A pivotal role of energy metabolism has been suggested in various neurological disorders including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, tardive dyskinesia and epilepsy. Although sophisticated noninvasive procedures are used to determine energy status in human brain, biochemical assay of adenosine-5-triphosphate (ATP) continues to be the method of choice in experimental animal studies. The results of our study showed that the sensitivity of luciferase bioluminescence (FLB) test can be enhanced several folds by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract followed by an optimum dilution prior to assay reaction. The luminescence signal remained stable for longer time when the test was performed in perchloric acid whereas a rapid decline in the signal intensity was observed when sterile water or Tris-EDTA were used as extractants.
Glial fibrillary acidic protein (GFAP) is a major intermediate filament protein of astrocytes and has been regarded as a sensitive and reliable marker of reactive gliosis in response to noxious stimuli. Reverse transcription polymerase chain reaction (RT-PCR) is regarded as the most sensitive and specific technique for measuring gene expression at mRNA level that can precisely predict the status of the respective protein. We have developed a sensitive method for estimation of low abundance mRNA using capillary electrophoresis coupled with laser fluorescence detection. This method is 100-fold more sensitive than conventional agarose gel electrophoresis and has been used to determine MPTP-induced increase in GFAP mRNA in midbrain of mice.
Capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF) is a highly sensitive, rapid and reproducible technique for a wide rang of applications including genotyping. We have demonstrated the superiority of fluorescence dye labeled nucleotides, [F]-dNTP-[R6G] over [F]-dNTP-[R110] for sensitive and reliable genotyping of the HLA-C gene using CE-LIF without necessitating any post-PCR cleanup.
Biogenic polyamines are sensitive markers for various diseases including cancer and urea cycle disorders. Polyamines are difficult to analyze by chromatography due to their high polarity and water-solubility. Proper derivatization of polyamines is an essential step for their chromatographic analysis. Now we have explored the chromatographic potential of CaSO4 for the analysis of aliphatic polyamines without attempting their derivatization. We have shown that CaSO4 is superior to silica gel for the separation of underivatized polyamines. The development time of CaSO4 plates was also about one-third shorter as compared to silica gel coated plates. This procedure was applied for the quantitative separation of polyamines in human urine samples. This is the first study reporting a TLC methodology for the separation of underivatized polyamines.