Chemistry: analytical and immunological testing – Metal or metal containing – Mn – te – re – fe – ru – os – co – rh – ir – ni – pd – pt
Reexamination Certificate
2000-10-05
2002-07-09
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Metal or metal containing
Mn, te, re, fe, ru, os, co, rh, ir, ni, pd, pt
C436S093000, C436S096000, C422S067000
Reexamination Certificate
active
06417006
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an assay method for detecting potential cardiovascular disease (CVD) in a vascularized subject, e.g. a human or non-human animal, especially a mammal, and in particular to an assay method which may be used to detect potential cardiovascular disease before the onset of CVD symptoms noticeable by the subject.
Cardiovascular disease is a major source of ill health among the human, population yet early or preemptive treatment, e.g. with change of diet, reduction or cessation of smoking, increase in regular exercise, prescription of lipid lowering drugs, etc., has a high success rate.
There is accordingly a need for methods which can be used to detect CVD or the potential for or propensity to CVD before the disease has progressed beyond the stage where treatment is routinely successful or may be used to prevent further progression of the disease, and in particular to detect CVD at the early stages when the symptoms are, not apparent to the patient or his physician.
Such methods may be used to screen the general population, or at-risk groups within the population, e.g. males over 40, workers in high stress jobs, patients with unhealthy diets, smokers, etc. where potential CVD or propensity to CVD is diagnosed, preemptive treatment may be given and/or the patient may be encouraged to make adjustments to lifestyle and habits. Likewise, where CVD, potential CVD or propensity to CVD is detected, the patient may be submitted to further testing, e.g. using more expensive or time consuming techniques, such as ECG, with and. without physical activity, radioisotope imaging of myocardial perfusion, X-ray (e.g. CT) myocardial angiography, MR myocardial angiography or perfusion imaging, etc. to confirm the presence and status of CVD. By the use of such assay methods as a “coarse filter” screening technique, unnecessary use of such expensive and time-consuming tests may be avoided while still increasing the. likelihood of as-yet undiscovered CVD being found and treated before health damage becomes irreversible.
SUMMARY OF THE INVENTION
The present invention is based on the realization that the protein complex holo-transcobalamin II, (holo TCII) a complex of the carrier protein transcobalamin II (TCII) and Vitamin B
12
(cobalamin) is at efficient marker for cardiovascular disease, and in particular that abnormal low holo-TCII levels in body fluids such as blood is indicative of CVD or susceptibility to CVD.
For the avoidance of doubt, the term “cobalamin” is used herein synonymously with “vitamin B
12
” and includes all forms of vitamin B
12
(e.g. cyanocobalamin; 5-6-dimethyl-benzimidazolyl cyanocobamide; methylcobalamine; 5′-deoxyadenoaylcobalamin) as may occur and be metabolically active (when appropriately presented) in the body.
DETAILED DESCRIPTION OF THE INVENTION
Vitamin B
12
(cobalamin) is a water soluble vitamin which forms part of the vitamin B complex found in foods. The core molecule consists of a corrin ring of four pyrole units which surround the essential cobalt atom. Cobalamin is the only vitamin which cannot be synthesised by animals or plants and must be absorbed from food in the gut. It can however be stored in the liver. It is synthesised by micro-organisms, in particular by anaerobic bacteria and yeasts.
Cobalamin functions in vivo as a co-enzyme and cobalamin enzymes catalyse three types of reaction: (i) intra-molecular rearrangements; (ii) methylations; and (iii) reduction of ribonucleotides to deoxyribonucleotides in some micro-organisms. In mammals, only two enzymic reactions, namely (i) and (ii) above, are known to require cobalamin as a co-enzyme.
In the process of digestion, a salivary protein called haptocorrin (which is also referred to in the art as R-binder or transcobalamins I and III collectively) binds cobalamin in the upper gastrointestinal tract forming a complex which passes through the stomach. Pancreatic enzymes digest the cobalamin-haptocorrin complex in the ileum, liberating cobalamin which is then bound to a protein called intrinsic factor, which is secreted by the gastric mucosa, to form a further complex. The cobalamin-intrinsic factor complex binds to a specific receptor in the lining of the terminal ileum, whereupon it is dissociated by a releasing factor and the cobalamin is transported actively across the membrane of the ileum into the blood stream.
Cobalamin does not circulate in the body in a free form in any appreciable amount. Probably 99% or so of cobalamin is bound by one of the transcobalamin proteins (TC I, II and III) or albumin.
The protein believed to be solely responsible for transporting cobalamin to target tissues is transcobalamin II (TCII), a critical trace protein without which cobalamin cannot cross cell membranes. Despite this important metabolic function, only about 6-25% of cobalamin in the serum is bound to TCII—most is carried by haptocorrin. TCII comprises a single chain polypeptide of about 40 kDa found primarily in serum, seminal fluid and cerebro-spinal fluid. Cobalamin bound TCII (i.e. holo-TCII) attaches to specific receptors on cell membranes and, once bound, the holo-TCII is taken into cells by pinocytosis. The holo-TCII constitutes the metabolically active pool of cobalamin, since none of the other cobalamin binding proteins, including transcobalamins I and III, are able to facilitate entry of the vitamin into cells.
TCII is synthesised by the liver, vascular endothelium, enterocytes, macrophages and fibroblasts and circulates predominantly as apo-TCII, i.e. lacking bound cobalamin. It has a short half life of approximately 90 minutes.
Less than about a quarter of the total plasma cobalamin is associated with TCII. The rest is bound to the other transcobalamins or albumin as mentioned above. The function or role of the non-TCII transcobalamins is unclear, but since they bind both cobalamin and cobalamin-like substances, they may play a role in ensuring that potentially harmful analogues of cobalamin cannot compete with cobalamin by virtue of them being unable to enter cells if bound to TC I or III. They may play a role in removing cobalamin analogues from the circulation or may serve as a store of cobalamins. Alternatively, they may ensure that free cobalamin and analogues thereof are not available for utilisation by micro-organisms.
Thus viewed from one aspect the invention provides an assay method for the detection of cardiovascular disease (CVD), potential cardiovascular disease, or propensity to cardiovascular disease in a human or non-human animal subject, said method comprising assessing the concentration of holo-transcobolamin II (holo TCII) in a cobalamin containing sample from said subject, e.g. a sample of blood, plasma, serum, seminal fluid, amniotic fluid or cerebrospinal fluid, preferably a sample of blood, plasma or serum, in particular a sample of serum.
By assessing it is meant that a quantitative or semi-quantitative value for the concentration of holo-TCII is determined. This may be a value for the concentration of the sample as tested, e.g. after treatment to remove cells or other sample components not being assayed for, or to concentrate or dilute the sample or to transfer the holo-TCII to a separate medium, e.g. a solid substrate.
Alternatively, the assessment may simply be qualitative, ie. to indicate whether the holo-TCII concentration is above or below one or more pre-selected threshold values, e.g. values indicative of absence of CVD detectable by the assay, presence of CVD (or potential CVD or propensity to CVD) as detectable by the assay, or uncertainty as to presence or absence of CVD, etc. The precise values for such threshold values or other reference values for holo-TCII concentration may depend on the nature of the sample, the age, weight, sex and species of the subject and may be determined in a routine manner by testing equivalent subjects without CVD or with CVD at various stages of development.
A value indicative of holo-TCII concentration determined (or “assessed”) in accordance with the method of the invention
Axis Shield ASA
Liniak, Berenato, Longacre & White LLC
Orzechowski Karen Lee
Wallenhorst Maureen M.
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