Bone and mineral metabolism
Bone is a special connective tissue hardened by mineralization with calcium phosphate in the form of hydroxyapatite [Ca5(PO4)3(OH)2]. Bone is composed of approximately 70% mineral and 30% organic matter (cells, collagen and non-collagenous proteins).
Bone has important functions, because it provides rigidity and shape, protection and support for body structures, and aids locomotion. Bone tissue is a dynamic structure undergoing constant remodeling. The bone turnover allows the bone tissue to repair itself and to adapt to forces placed on it. During childhood, bone turnover is very high and formation outweighs resorption. In young adulthood, formation and resorption are in equilibrium, but after age 35, there is a net loss. The bone’s overall mechanical properties are a combination of the rate of bone turnover, collagen matrix, structure, size, geometry, and the bone density. In order for the strength of the bone to be maintained, the bone turnover must be carefully regulated .
- The cortical or compact bone, which comprises up to 85% of the bone mass has a dense ordered structure. Cortical bone is found mainly in the shaft of long bones and the surfaces of flat bones. It is composed of bone laid down concentrically around central canals (Haversian systems). The canals contain blood vessels, lymph vessels, nerves and connective tissue. A concentric layer of rings or lamellae of bone matrix surround each Haversian canal. Tiny spaces (lacunae) containing osteocytes are within the lamellae.
- The cancellous or trabecular bone is lighter than the cortical bone and has an irregular structure. Trabecular bone forms the ends of long bones and the inner parts of flat bones. It contains interconnecting plates and bars called trabeculae, with intervening marrow lending it a honeycomb appearance. The trabeculae are aligned along lines of stress; this connectivity adds considerably to its strength. The collagen fibres are parallel to one another arranged.
In general, each bone has an outer layer of cortical bone overlying trabecular bone and the medullary cavity . The cortical bone has an outer membrane called the periosteum. The periosteum has two layers:
- The outer fibrous layer
- The inner cell layer with osteogenic potential which lays down new bone allowing the bone to enlarge, a process known as periosteal apposition.
The inner surface of the cortex has another lining called the endosteum. Bone tends to undergo resorption from the endosteal surface.
Both the periosteum and the endosteum contain the multicellular unit of bone which comprises osteocytes, osteoblasts and osteoclasts and their precursors. Osteoblasts and osteoclasts function in a coordinated manner, by their respective bone forming and resorbing activities, to carryout remodeling, growth and repair .
Bone turnover is a dynamic process, which constantly renews the bone and maintains its mechanical competence. Each year, 5–10 % of the bone of the skeletal system is renewed in cycles. The term modeling is restricted to the period of skeletal growth, when the size and shape of the bone is determined. The process of bone turnover in adults is known as remodeling. Bone remodeling is part of the body’s way to manage mineral (i.e., calcium and phosphate) homeostasis, and a mechanism for repair and adaption . The basic multi-cellular unit (Bone Remodeling Unit, or BRU) of remodeling, comprises the osteocytes, osteoblasts, and osteoclasts. The activity of the BRUs is regulated by mechanical forces, bone cell turnover, hormones (e.g., parathyroid hormone, growth hormone, calcitonin) and local factors . The remodeling is partially initiated by the osteocytes (), which detect mechanical stress and respond to biochemical stimuli. Activation results in the lining cells of the endosteal surface being retracted, and digestion by matrix metalloproteinases of the endosteal membrane . Osteoclasts are then recruited, followed by fusion of activated osteoclasts to become multi-nucleated osteoclasts . The activated osteoclasts cause resorption of the underlying bone forming a resorption cavity. The activation and resorption process takes 10–15 days. In the next step osteoblasts are recruited to the cavity, lay down new osteoid which becomes calcified. The entire process is completed after 3–6 months. The rate of bone turnover is dependent on the type of bone, being highest in sites where trabecular bone predominates (vertebrae) and lowest in cortical bone (hip) .
During skeletal development osteoblast differentiation and bone matrix deposition occurs. The spatial and chronological coordination for this involves the interaction of endocrine, paracrine and autocrine factors. Included are the epidermal growth factor (EGF), the osteoblast-stimulating factor-1 (OSF-1), parathyroid hormone, growth hormone, prostaglandin and insulin-like growth factors (). The development of the skeleton requires the chronologically and spatially controlled molecular interaction between Wnt/catenin and transforming growth factor-β/BMP bone morphogenetic protein mediated signaling pathways . The expression of Runx2 is required for multi potent stem cells to differentiate into osteoblastic lineage . Runx 2 is a key osteoblast differentiation transcription factor. A suppressing effect on the Wnt/β-catenin signal pathway has the Dickkopf-1 protein. Other details are listed in .
Osteoblasts, encased by the matrix that they themselves synthesize become osteocytes, terminally differentiated cells. Osteocytes have less matrix forming activity, and the alkaline phosphatase activity and basophilic nature of its cytoplasm is reduced. The cells occupy small lacunae within the bone matrix and constitute more than 90% of the bone cells. Osteocytes, isolated by bone matrix from each other communicate through protoplasmic extensions via lacuno-canaliculi with neighboring cells. These processes may have the potential to stimulate bone resorption. Osteocytes act as mechanosensory cells and are responsible for the maintenance of bone structure and mass. By far the most abundant matrix protein in the osteocyte environment is type-1 collagen. The phenotype of the osteocyte and the formation of osteocyte processes depend on cleavage of type-1 collagen .
Osteoclasts are large end-differentiated multi nucleated cells derived from bone marrow macrophages of the hematopoietic lineage /, /. The unique function is resorbing bone matrix. Osteoclastogenesis is dependent on cytokines generated in the bone micro environment. Osteoblasts control osteoclast formation through the expression of cytokines like RANKL (receptor activator of nuclear factor-kB ligand) and M-CSF (monocyte-colony stimulation factor). M-CSF is important for the proliferation of osteoclast progenitors. RANKL controls the differentiation process by activating the RANK receptor, the osteoclast associated receptor (OSCAR) and the triggering receptor expressed on myeloid cells-2 (TREM-2). In contrast to this, osteoprotegerin, a receptor of the tumor necrosis factor receptor superfamily, suppresses the formation of osteoclasts (). Additional details on this are listed in .
Osteoclast formation is also controlled by circulating hormones like parathyroid hormone, calcitriol, trans growth factor-β and estrogens.
The osteoclastic bone resorption is important for modeling and remodeling the bone and for the calcium homeostasis.
Normal bone physiology depends on the interaction of osteoblasts and osteoclasts. There are, however, diseases in which this interaction is disrupted, such as osteoporosis, Paget’s disease, inflammatory arthritis, as well as bone tumors and bone metastases.
- 90% collagen type 1
- 5% proteoglycans (chondroitin sulfate, glucuronic acid, heparan sulfate, decorin, biglycan, versican), cell adhesion molecules (fibronectin, thrombospondin, vitronectin, osteopontin, bone sialoprotein), γ-carboxylated proteins (1% osteocalcin, matrix Gla protein, protein S)
- 3% growth factors and 2% osteonectin.
OPN, also known as secreted phosphoprotein-1, is a large phosphoglycoprotein profibrotic adhesion molecule with several domains, one of which has binding affinities for integrins (e.g., the vitronectin receptor) . The molecule is attacked at several points by thrombin and matrix metalloproteases, resulting in fragments which either promote or reduce cellular adhesion. OPN binds CD44, which interacts with the ERM (ezrin, radixin, moesin) family of adapter proteins that link to actin in the cytoskeleton.
Bone sialoprotein (BSP)
BSP is a cell adhesion molecule produced by osteoblasts and is stored in the organic bone matrix. BSP is part of the non-collagen connective tissue matrix of bones, dentin and calcifying cartilage. If BSP is increased in serum, this can best be correlated with bone-resorptive processes . Since thrombocytes also contain BSP, changes in the thrombocyte count also affect the BSP concentration in serum.
Active osteoblasts synthesize the bone matrix which is composed of organic (osteoid) and inorganic (mineral) components . The osteoid consists predominantly of type-1 collagen, with small amounts of proteoglycan, lipids and several non collagenous proteins (osteocalcin, fibronectin, osteonectin and beta-carboxyglutamic acid containing protein). The osteoid comprises about one third of the total skeleton weight.
The inorganic mineral phase consists of crystals of hydroxyl apatite. The crystal is small and in the order of 200 by 30 to 70 Angstroem. The mineralization of the osteoid takes place via the osteoblastic exocytotic release of vesicles, in which the hydroxyapatite nucleation takes place in the matrix. Together with the matrix components of the osteoid, crystallized matrix vesicles form the bone matrix outside the osteoblasts . The speed of the of the hydroxyapatite crystallization from amorphous calcium phosphate is controlled by matrix components. The resorptive cavity created by the osteoclast is often the site of subsequent osteoblastic activity which fills the cavity with new bone.
In lamellar bone, the mineralization appears to occur directly mediated by the lining osteoblasts without the exocytotic release of vesicles.
During adulthood, bone is remodeled in order to adapt acute changes in calcium homeostasis, bio mechanical demands and to renew and repair old bone substance and osseous micro fractures. Hormones that systemically affect the bone metabolism are growth hormone/IGF-1, estrogens and testosterone (), glucocorticoids, thyroid hormone, parathyroid hormone and 1,25(OH)2D. See ).
In childhood, GH promotes skeletal growth via a direct effect on the mature chondrocytes of the epiphyseal plate (i.e., secretion of insulin-like growth factor 1 (IGF-1)) by these cells is increased. IGF-1 together with transforming growth factor-α (TGF-α), which is also produced by chondrocytes, exerts a mitogenic effect (in a paracrine manner) on the chondroblasts and promotes their differentiation with a resultant expansion of the cartilage layer in the epiphyseal growth plate. Preosteoblasts and preosteoclasts also have GH receptors. The cell proliferation and differentiated cell functions of osteoblasts (e.g., the synthesis of collagen type 1 and the excretion of IGF-1) are positively influenced by GH. In the adult skeleton, GH/IGF-1 thus maintains both the processes that are required for the continuous remodeling of bone tissue (i.e., both formation as well as the resorption of bone) .
The osteoprotective effect of estradiol on bone metabolism is well established. Estrogens influence osteoblasts and osteoclasts via two estrogen receptors (ER). The ERα is an activator and the ERβ is an inhibitor of the estrogen effect, because ERβ uses the ERα to form a heterodimer and thus deactivates it. The relative expression of both ERs on the bone cell decides its estrogen response. The density of ERα is greater in the developing compact bone type and of ERβ in the cancellous (trabecular) type. In estradiol deficiency, the activity of the osteoclasts is higher than of the osteoblasts with the net effect of osteopenia .
Estrogen deficiency is the most important factor in the pathogenesis of the post menopausal bone loss. Post menopausal women with estradiol values of > 10 ng/L have a normal bone fracture rate, with values of ≤ 10 ng/L a moderate increased rate and with < 5 ng/L a strong increased rate, especially in the spinal column. If, several years after menopause, a low dosage of transdermal estrogen is supplied (tape with 25 μg estradiol release per day), the serum value of estradiol maintains at about 20 ng/L. The benefit for the patient is an increase of bone mineral density (8% in 3 years), especially in the spine, which can scarcely be improved upon even by increasing the estradiol dosage (9% in 3 years with 50 μg estradiol release per day) .
Hormonal contraceptives do not cause an increased risk of bone fracture, but they can disturb the development of peak bone mass if they are taken within the first 3 years after menarche . New investigations show that cyclic variations in FSH levels had a positive effect on the concentration profile of the bone resorption marker β-CTX in women . FSH stimulates osteoclast formation by activating osteoclast-bound FSH receptors.
The supply of estrogen is also important for men in suspicion of developing osteoporosis, because they also have osteoblast and osteoclast estrogen receptors. Osteoporosis is cumulatively diagnosed for an estradiol concentration below 13 ng/L .
Androgens stimulate the proliferation and differentiation of osteoblasts both directly and via the GH/IGF-1 axis. The signal is transmitted via androgen receptors, which are present in low densities on the osteoblasts. The effects of androgen are especially evident from puberty on when achieving the maximum bone mass of the skeleton, which depends on timely excretion of androgens. Thus, delayed puberty in men is associated with reduced bone mass . Androgens decrease bone resorption of osteoclasts indirectly by inhibition the recruiting of osteoclast precursor cells. This happens due to reduced secretion of IL-6 and prostaglandin E2 by osteoblasts and stromal cells.
In addition to the androgen receptor, bone cells also have 5α-reductase activity, which is responsible for the conversion of testosterone to dihydrotestosterone (DHT). DHT is the most important biologically active metabolite of testosterone in the tissues. Testosterone can also be aromatized to estradiol catalyzed by aromatase; bone cells have aromatase activity.
Similar to ovarectomy, orchi ectomy will lead to increased bone resorption and rapid bone loss.
Physiological plasma glucocorticoid concentrations promote important osteoblastic functions (type I collagen synthesis, ALP synthesis) and simultaneously suppress the differentiation of osteoclast precursor cells into active osteoclasts. Therefore, physiological glucocorticoid concentrations exert a stabilizing effect one bone metabolism without any negative effects on calcium homeostasis .
High glucocorticoid concentrations over a prolonged period of time have an unfavorable effect on calcium homeostasis. The cancellous bones are especially affected. After glucocorticoid treatment is started, there is a rapid phase of bone loss, followed by a slower phase of demineralization. The extent depends on the dosage and the duration of the treatment. However, there are also patients with normal bone density who undergo glucocorticoid treatment. In patients with Cushing’s syndrome, fractures are more frequent the longer the disease persists.
Physiological thyroid hormone concentrations promote bone turnover. This is caused by stimulation of osteogenic cells as mediated by the osteoblast triiodothyronine (T3) receptor. As a result of stimulation, these cells produce signal peptides which exert a positive effect on osteoclast differentiation . Hence, bone resorption increases although no net loss in bone mass occurs because of the coupled new bone formation and the T3 induced stimulation of specialized osteoblast functions.
In the case of permanently increased thyroid hormone concentrations, the activation of osteoclasts may exceed the bone-producing capacity of the coupled bone formation since T3 also exerts an inhibitory effect on osteoblast proliferation; consequently, a loss in bone mass can occur. Juvenile hypothyroidism causes stunted growth.
Bone is subject to constant remodeling in adulthood:
- To adapt to acute changes in calcium homeostasis
- To adapt to bio mechanical stress
- To renew and repair old bone substance and bones with micro fractures.
The bone metabolism needs sufficient calcium supply from the diet and fluid to maintain these functions. The absorption of calcium can fluctuate between 25% and 70% of calcium in the diet, depending on the calcium content and components (complexing agent, pH) of the food and the presence of gastrointestinal disorders. Small changes in the plasma calcium concentration due to calcium malnourishment, vomiting, diarrhea, and malabsorption are registered by the parathyroid cells and are responded with an increased secretion parathyroid hormone (PTH) and of 1,25 (OH)2D (calcitriol). Calcium in bone is mobilized with a consecutive increase in the serum calcium level.
- PTH increases the number and the resorptive capacity of the osteoclasts. PTH also suppresses the activity of the osteoblasts and thus prevents storage of calcium in the bones. The increase in osteoclast activity occurs indirectly via the PTH stimulation and directly due to the activation of the osteoclasts. The latter have PTH receptors like the osteoblasts.
- Mediated by calcitonin, because this hormone constitutes to the stabilization of the calcium concentration in plasma (see ). Its primary effect is the suppression of the osteoclast-conveyed bone resorption as a response to a systemic increase of the calcium level. Calcitonin mediates its effects via the adenylate cyclase pathway. In the small intestine, calcitonin promotes the transcellular absorption of calcium by increasing the calbindin expression of the enterocytes. In the kidneys calcitonin enhances the re absorptive capacity for calcium in the thick, ascending part of the loop of Henle.
In metabolic bone disease (Paget’s disease, rickets and osteomalacia, primary and secondary hyperparathyroidism) and osteoporosis biomarkers of bone metabolism and bone turnover are important clinical tools in patient management. The biomarkers are supplementary to imaging procedures, bone mineral density measurements, and possible histomorphological examinations of bone aspirates. Routine investigations and bone markers for the diagnosis and monitoring of bone disorders are shown in .
Bone turnover markers reflect whole body rates of bone resorption and bone formation. The markers provide a dynamic assessment of the skeleton and can provide real-time assessment of bone remodeling. Bone turnover markers are differentiated in bone resorption and bone formation markers .
- Pyridinoline (PYD) and deoxypyridinoline (DPD) in the morning urine (see ).
- Cross-linked carboxy-terminal telopeptide of type I collagen (CTX) in blood (see ).
The most important indication for bone resorption markers such as CTX, PYD and DPD is monitoring the course and assessment of the progress in treatment of osteoporosis. The markers give information:
- Whether a high turnover bone metabolism exists
- How high the extent of the bone resorption is
- Whether there is a response to treatment.
Within the scope of monitoring osteoporosis, the optimal points in time of the response to treatment with bisphosphonates is 1 month after the start of the treatment and 6 months for estradiol substitution.
Bone resorption markers are also indicated:
- For osteomalacia/rickets
- For monitoring of Paget’s disease
- For estimating the bone participation in hyperparathyroidism.
In the serum, the markers that are preferably measured are those which show greater activation of the osteoblasts than the osteoclasts:
- Alkaline phosphatase, especially the bone isoform (bone ALP). Thus, according to the KDIGO, the assay of the ALP or bone ALP in CKD patients in stages 3 to 5 is recommended annually for diagnosing CKD-MBD.
- Osteocalcin (see ).
- N-terminal pro peptide of type 1 collagen (P1NP).
These markers are important, especially the bone ALP in combination with the PTH assay, if adynamic bone disease is suspected in dialysis patients.
Tables 6.1-5 to 6.1-14 list laboratory findings in physiological conditions and for diagnosing, differentiating and monitoring bone diseases:
19. Garnero P, Delmas PD. Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. J Clin Endocrinol Metab 1993; 77: 1046–53.
21. Vesper HW, Demers LM, Eastell R, Garnero P, Kleerekoper M, Robins SP, et al. Assessment and recommendations on factors contributing to preanalytical variability of urinary pyridinoline and deoxypyrodinoline. Clin Chem 2002; 48: 220–35.
30. Anlinker M, Bieglmayer C, Dimai HP, Gasser RW, Kudlacek S, Obermayer-Pietsch B, et al. Labordiagnostik in der Prävention, Differentialdiagnose und Verlaufskontrolle der Osteoporose. J Lab Med 2009; 33: 140–6.
31. Prince RL, Smith M, Dick IM, et al. Prevention of postmenopausal osteoporosis. Comparative study of exercise, calcium supplementation, and hormone replacement therapy. N Engl J Med 1991; 325: 1189–95.
46. Sullivan W, Carpenter T, Glorieux FH, Travers R, et al. A prospective trial of phosphate and 1,25-dihydroxyvitamin D3 therapy in symptomatic adults with X-Iinked hypophosphatemic rickets. J Clin Endocrinol Metab 1992; 75: 879–84.
58. Takai M, Ono J, Okamoto M, Fujimoto K, Kamei A, Nunomura S, et al. Establishment of a novel ELISA system for measuring periostin independently of formation of the IgA complex. Ann Clin Biochem 2022; 0 (0): 1–10.
The hormonal regulation of calcium metabolism as well as the interrelated phosphate metabolism is complex. Reciprocal relations between the small intestine, skeleton, kidneys and the endocrine system, in particular the parathyroids, maintain calcium and phosphate homeostasis.
Without the exception of tetany, clinical symptoms caused by calcium status disorders are often initially misinterpreted. Therefore, the diagnosis is often established incidentally during the routine determination of serum calcium and phosphate or if secondary changes, especially of the kidney and bone are present.
The determination of serum calcium and phosphate is recommended for early detection and differentiation of bone disorders and in chronic renal disease. The determination of parathyroid hormone is an important follow-up investigation of hyper- and hypocalcemia.
The calcium consists of three fractions:
- Free or ionized calcium (iCa), it accounts for 50% of calcium
- Protein-bound calcium, most of which is bound to albumin with only a small portion bound to globulins; it accounts for 45% of calcium
- Complex bound calcium, the portion bound to anions, especially phosphate, citrate and bicarbonate accounts for 5% of calcium.
In routine diagnostics calcium is primarily determined in serum or plasma. The assay of iCa in anticoagulated whole blood or plasma is mostly requested secondarily to clarify hypocalcemia or, more rarely, hypercalcemia.
Under routine diagnostic conditions the measurement of calcium is easier than that of iCa, but has the disadvantage that serum calcium is greatly influenced by the concentration of protein, especially albumin. Because albumin is responsible for 80–90% of the protein-bound calcium fraction. Accordingly, a decrease in albumin by 10 g/L results in a decline of serum calcium by 1 mg/dL (0.25 mmol/L).
Ionized Ca (iCa)
The iCa is an indicator of the biologically active calcium, because its plasma concentration is directly regulated by PTH and 1,25(OH)2D. Therefore, iCa responds sensitively to disturbances in calcium homeostasis, but it cannot be determined in every laboratory. The iCa is influenced by pH changes. A shift of the pH of 0.1 in either direction causes an inverse iCa change of 0.05 mmol/L. Pre-analytical sources of errors, which cause a pH shift in vitro, must therefore be taken into consideration. The most important biological causes which cause a change in pH in vitro are:
- The loss of CO2 from the specimen into the surrounding air. Due to solution of CO2 in blood there is a rise in pH with a consecutive decrease of iCa. The sample must therefore collected under anaerobic conditions. The sample tubes must be completely filled and the sample must be hermetically sealed.
- Formation of lactate in the specimen due to glycolysis. As a result, the pH decreases and the concentration of iCa increases. This can be prevented by the rapid separation of the blood cells from the plasma or by the addition of glycolysis inhibitors such as sodium fluoride.
Screening: every 2 years after age 50, including recording height and weight (concern: osteoporosis; a decline in height by more than 1 cm every 2 years necessitates further examinations).
Critically ill patients: intensive-care patients, surgical patients during and after major operations.
Newborns: in the event of premature delivery, maternal diabetes, asphyxia, hypo tension, cramps, hypoglycemia, sepsis.
Tetanic syndrome: investigation of the hypocalcemic form.
Bone: spontaneous fractures, osteoporotic fractures (femoral neck, spine, radius), bone pain, radiographically detected bone changes, dental changes, growth disorders.
Kidney: nephro- or urolithiasis, nephro calcinosis, polydipsia, polyuria, chronic kidney disease, dialysis patients.
Neuromuscular: tetany, seizures, suspicion of hypoparathyroidism after thyroid surgery, headache, muscular weakness.
Parathyroids: suspicion of hyperparathyroidism, hypoparathyroidism and pseudohypoparathyroidism.
Genetic disorder: suspicion of autosomally dominant hypocalcemia.
Psyche: fatigue, loss of drive, lethargy, depression, anorexia.
Stomach and intestines: peptic ulcer disease, pancreatitis, gall stones, recurrent diarrhea, malabsorption, obstipation.
Skin and skin appendages: changes involving the skin, nails, hair, hyperpigmentation of skin.
Lung: sarcoidosis, tuberculosis, other granulomatous diseases.
Tumor: weight loss, malignoma, lymphoma, treatment with cytostatics, radiotherapy.
Endocrine: thyroid, testicular, ovarian, adrenocortical diseases.
Medications: intake of vitamin D and its metabolites or analogues, vitamin A, anti-epileptic drugs, corticosteroids, thiazides, digitalis.
Total calcium is easier to determine than ionized calcium in clinical routines.
Atomic absorption spectroscopy (AAS)
Principle: in AAS calcium is not excited in the flame but dissociated from its chemical bonds and placed in an unexcited ground state as neutral atom. The neutral atom has low energy level and is capable of absorbing radiation corresponding to its own line spectrum. Energy of a line specific spectrum is absorbed when atoms with the same line-specific spectrum absorb radiation. In this way, AAS is the converse of the emission procedure for determining metals e.g., flame spectrophotometry. The components of the AAS are:
- Hollow cathode lamp; this generates the element-specific linear spectrum and is different for the individual element
- A gas flame or electrically heated graphite furnace cuvette for specimen atomization at high temperature
- Photomultiplier that transforms the decrease of the light from the hollow cathode lamp, when a proportion is absorbed from the ground-state atoms in the flame, into an electrical signal.
In AAS, the absorption change measured at the signal display unit is proportional to the concentration of the element in the specimen.
Principle: in flame emission photometry the specimen to be measured is diluted with A. bidest and exited by an acetylene gas flame. Light is emitted at a specific wavelength as calcium returns to its lower energy level. Due to a partial overlap of the emission bands of sodium and calcium at 622 nm, a compensation solution which contains a sodium concentration that is identical to that of normal serum is used to compensate the emission of sodium.
Principle: calcium forms chromophores with the metal complexing dyes o-cresolphthalein complexone and arsenazo III . At pH 10–12, the reaction of o-cresolphthalein complexone with calcium forms a red complex, which is measured at 570–575 nm. The complex is stabilized by adding KCN; this also eliminates interferences caused by heavy metals. Interference due to magnesium ions is prevented by the presence of 8-hydroxyquinoline.
Principle: the concentration of iCa is measured using an electrode arrangement, which consists of a flow-through calcium-selective electrode and an Ag/AgCl reference electrode. An ion-exchanger as the active phase of the measurement electrode is separated from the specimen to be analyzed by a porous membrane and within the electrode it is in contact with a millimolar CaCl2solution; an electrode immersed in this solution leads the electrical potential to a measuring device. When the specimen flows through the calcium-selective electrode, the iCa reacts with the ion-exchanger.
By moving charged particles over the porous membrane, the active electrode phase becomes increasingly charged compared to the serum. A difference in potential forms, which is measured against the constant potential of the reference electrode. Between the potential recorded at the measuring device and the logarithm of iCa activity, there is a linear relation.
The measurement of iCa is influenced by the pH value of the blood, because iCa and H+ ions compete for protein binding sites. When the pH increases, the concentration of iCa decreases. Some analyzers simultaneously measure iCa and pH and correct the iCa to pH 7.4 for samples with a changed pH value. This is not recommended because 70% of the patients with low iCa are not detected .
Serum, plasma (ammonium heparinate): 1 mL
- Whole blood (calcium heparinate), the measurement should be carried out within 40 min. after sampling
- Serum or plasma (Ca-saturated heparin) if centrifugation and measurement are done immediately after sampling
- Serum or plasma (Ca-saturated heparin). Sampling in vials with separator gel and then centrifugation if measurement is not earlier possible as 24–72 hours after sampling.
Calcium (corr.) mmol/L= Total calcium (measured) mmol/L – 0.025 × albumin (g/L) + 1.0
Albumin-adjusted calcium is determined in patients with dysproteinemia.
The suggested calcium intake for people without kidney disease is 1,000–1,300 mg per day (25 to 32 mmol).
The calcium metabolism can be assessed based on total calcium or iCa. Approximately 40% of total calcium is bound to albumin, but only the iCa is biologically active in the extracellular fluids. The total calcium level is clinically equivalent to the iCa if a change in protein concentration and dysproteinemia are ruled out.
- For critically ill patients and if an intravenous calcium substitution is considered
- If hypoproteinemia or dysproteinemia is present (e.g., in pregnant women, newborns, renal or intestinal protein loss, malabsorption syndrome, multiple myeloma, chronic inflammation)
- In the final stage of chronic kidney disease
- In cases of hypercalcemia, especially if PTH is normal and there are no signs of tumor
- To verify hypocalcemia postoperatively or intraoperatively (thyroid and parathyroid operation, open heart surgery, liver transplantation)
- If there is a massive transfusion of citrated blood or of fresh frozen plasma
- In cases of increased PTH and normal calcium as a result of a vitamin D deficiency, insufficient enteral calcium absorption or hypercalciuria.
According to a study approximately 72–76% of the iCa measurements can be spared if they are only performed in cases with total calcium levels below 8 mg/dL (2.0 mmol/L), because patients with clinically relevant iCa levels below 4 mg/dL (1.0 mmol/L) will still be detected.
If it is not possible to measure iCa, the calcium can be corrected to an albumin value of 40 g/L (recommended for dialysis patients) or a protein value of 77.6 g/L for a better assessment of hypocalcemia when there is a decreased concentration of albumin or total protein (). This applies to children older than 1 year and to adults.However, Ca correction for hypoalbuminemia is not an accurate concept. Studies have shown that in patients with endocrine disorders or acid-base disturbances, patients who are critically ill, and patients undergoing hemodialysis the correction formula is not an acceptable substitute for iCa .
The reference intervals for total calcium vary in literature by up to 1.6 mg/dL (0.40 mmol/L). The following is generally accepted for adults: a mean value of 9.4–9.5 mg/dL (2.35–2.38 mmol/L), an upper reference interval value of 10.5 mg/dL (2.62 mmol/L), and a lower reference interval value of 8.6–8.8 mg/dL (2.15–2.20 mmol/L).
The calcium concentration in serum is kept constant in a narrow range under the control of PTH. The relationship between iCa and PTH is transmitted via the calcium-sensitive receptor. This is expressed on the cell membrane of parathyroid cells and renal tubular cells, where it regulates the calcium reabsorption. Individual activating or deactivating mutations of the calcium-sensitive receptor genes can cause hypercalcemic or hypocalcemic disorders such as the familial hypocalciuric hypercalcemia (FHH), the neonatal severe primary hyperparathyroidism (NSHPT), or the autosomally dominant hypocalcemia with hypercalciuria (ADHH) .
The authors of a recently published paper concluded that ionized calcium, measured in heparinized whole blood collected anaerobically into blood gas syringes, would appear as the more reliable parameter for assessing calcium status in hospitalized patients.
If there is an increase in total calcium above 11.2 mg/dL (2.8 mmol/L) and rising values, organ systems can be negatively impacted. Since the effect of the antidiuretic hormone and the renal sodium transport are suppressed, the renal concentrating capacity is reduced. The result is sodium excretion with polyuria, exsiccation and polydipsia. The symptoms of chronic hypercalcemia are fatigue, depression, myasthenia, abdominal pain and bone pain.
In hypercalcemic syndrome a life-threatening crisis with values of more than 14 mg/dL (3.5 mmol/L) has developed due to dehydration. The consequences are limited consciousness up to a coma, exsiccosis, hyperpyrexia, oliguria preceded by polyuria, arrhythmia, encephalopathic symptoms, and bradyarrhythmias.
In more than 90% of cases with hypercalcemia, there is a clinically manifest disease or the hypercalcemia can be further differentiated by biochemical investigations. A small proportion of the remaining causes are related to medications. Thus, in one study , in which two serum calcium values > 10.6 mg/dL (2.64 mmol/L) or at least one value ≥ 10.8 mg/dL (2,70 mmol/L) was the criterion, the underlying cause of hypercalcemia was in 46% of the cases a malignoma and in 35% the primary hyperparathyroidism. In the remaining 19% of the cases, the hypercalcemia was connected with the medication of thiazides, an increased 1,25 (OH)2D concentration or immobilization .
Hypercalcemias result from the interaction of several mechanisms, such as increased intestinal absorption, increased bone resorption, exsiccosis, and reduced renal calcium excretion.
If the hypercalcemia is only temporary, the examinations must be repeated. In that case, preferably iCa should be determined in addition. Pseudo-hypercalcemia due to a hemoconcentration, often caused by a long period of venous occlusion when drawing blood, must be taken into consideration.
The differentiation of an unclear hypercalcemia can be possible by determining parathyroid hormone (PTH) level . With few exceptions, the concentration of PTH allows the differentiation of the two most common causes of hypercalcemia (e.g., primary hyperparathyroidism (pHPT) and tumor hypercalcemia).
Increased PTH: if PTH has been increased to more than 30% of the upper reference interval value, this most likely indicates pHPT. Some rare causes are lithium-induced hypercalcemia, tertiary hyperparathyroidism, and ectopic PTH formation.
PTH slightly increased or near the upper reference interval value: in such cases the calcium excretion in 24-hour urine should be determined. If the excretion is low, familial hypocalciuric hypercalcemia must be taken into consideration.
PTH normal or suppressed: the search for a malignant tumor should be the primary aim:
- If a tumor is diagnosed, the PTH-related peptide (PTHrP) should be determined to clarify the cause of the hypercalcemia. Increases indicate a paraneoplastic PTHrP formation which is often associated with adenocarcinoma of the lung, carcinoma in the head and neck area, of the pancreas, ovaries and the urogenital tract.
- If a granulomatous disease is present (sarcoidosis, tuberculosis, lymphoma), the 1,25(OH)2D formation in the granulomas is often the cause of the hypercalcemia. If the serum concentration of 1,25(OH)2D is normal multiple myeloma, bone metastases, immobilization, hyperthyroidism or medication may be the cause. Biochemical findings for diseases with hypercalcemia are shown in .
Up to 30% of cancer patients have hypercalcemia during the course of their disease. Cancer-associated hypercalcemia is a complication of advanced cancers. Patients, who received chemotherapy and had normalization of calcium concentration had longer survival. Hypercalcemia is most common in patients with breast cancer, non-small-cell lung cancer, squamous-cell cancers of the head and neck, multiple myeloma, ovarian cancer, and urothelial cancer. Increased osteoclastic bone resorption is responsible for hypercalcemia, regardless of tumor type. The use of bone-resorption inhibitors to lower calcium concentration is the pathobiochemical principle of treatment /, , /.
1. Humoral; hypercalcemia is caused by tumor secretion of parathyroid hormon related protein (PTHrp). Secretion of PTHrp is associated with squamous-cell cancers of the head and neck, breast carcinoma, urothelial carcinoma, and squamous tumors of the lung. The patients have no or few bone metastases.
2. Humoral in nature; the cancer cells produce hormones involved in bone remodelling e.g.,
- up-regulation of the expression of CYP27B1 (encoding 1-alpha-hydroxylase, the enzyme responsible for converting 25-hydroxyvitamin D to the active 1,25-dihydroxyvitamin D). Excess of 1,25-dihydroxyvitamin D leads to hypercalcemia by increasing intestinal calcium absorption as well as calcium resorption from bone.
- ectopic production of parathyroid hormone
3. Osteolytic; hypercalcemia results from extensive bone metastases, resulting from local action of tumor cells in the bone. Tumor cells secrete cytokines that act to increase osteoclastic bone resorption. Osteolytic hypercalcemia often results from mutiple myeloma or breast cancer.
- Low parathyroid (PTH) levels e.g., genetic disorders that result in abnormal PTH concentrations and acquired causes that include infection, autoimmune disorders (autoimmune polyglandular syndrome type 1), destruction of the parathroid gland (after surgery or radiation therapy)
- High PTH level e.g., vitamin D deficiency, PTH resistance, poor calcium intake or absorption, calcium loss.
Hypocalcemia is a status of serum calcium below 8.8 mg/dL (2.2 mmol/L) and of iCa below 4.0 mg/dL (1.0 mmol/L).
In cases of mild hypocalcemia, the calcium level is 8.0–8.7 mg/dL and iCa is 1.00–1.12 mmol/L.
In severe hypocalcemia, the calcium level is below 6.4 mg/dL and iCa is below 0.80 mmol/L. The calcium data applies in cases of normo proteinemia.
Hypocalcemia occurs physiologically in newborns in the first 3 days of life and is regarded as a natural stimulus for PTH secretion. The most frequent cause of a decreased calcium is hypoalbuminemia. Although in some of the cases iCa concentration is normal, hypoalbuminemia is often a symptom of diseases that are associated with a decrease in iCa. Examples for this are:
- Proteinurias with a loss of calcium-binding protein, that result in a deficiency of 25(OH)D
- Malabsorption syndrome, which also leads to a 25(OH)D deficiency.
In the presence of unclear hypocalcemia or hypoalbuminemia, preferably iCa can be determined separately. The measurement of calcium excretion in the 24-hour urine and of serum PTH is also important for differential diagnostics.
- PTH decreased; hypocalcemia is caused by a reduction of PTH-stimulated functions (i.e., the intestinal absorption of calcium, the mobilization of calcium from the bone, and the renal tubular reabsorption of calcium)
- Pseudohypoparathyroidism (PTH elevated) which is characterized by end-organ resistance to PTH; its manifestations are comparable to those seen in hypoparathyroidism. To distinguish pseudohypoparathyroidism from secondary hyperparathyroidism, it may be necessary to perform the Ellsworth-Howard test.
- Renal failure (PTH elevated); the following mechanisms are responsible for hypocalcemia:
a) renal phosphate retention if the glomerular filtration rate is less than 25 [ml × min–1 × (1.73 m2)–1] (PTH elevated)
b) decreased intestinal calcium absorption, caused by 1,25(OH)2D deficiency that in turn is a result of reduced 25-OHD-1α-hydroxylase activity which occurs as the mass of kidney tissue is reduced (PTH elevated)
c) resistance of the bone to PTH with reduced calcium release by osteoclasts (PTH elevated)
d) defects in the metabolism of vitamin D, and vitamin D deficiency (PTH elevated)
e) hypomagnesemia that results in decreased PTH secretion and end-organ resistance to PTH
f) hyperphosphatemia which causes 1,25(OH)2D deficiency because of the inhibition of 25-OHD-1α-hydroxylase (PTH elevated).
Hypocalcemias occur frequently in critically ill patients, especially in cases with sepsis, cardiac, renal or pulmonary failure, and after major surgery or serious burns. It may result from a shift of Ca to the intracellular compartment.
- If, in hypocalcemia, the cardiac function is to be improved by calcium infusions
- After open heart surgery, if the high cardioplegic calcium concentration has to be normalized
- During liver transplantation. Often many banked blood units containing citrate are administered and the serum calcium level can decrease below 2.0 mg/dL (0.5 mmol/L), because liver function can not metabolize the citrate which binds serum calcium.
- Hyperventilation which causes metabolic alkalosis and the clinical symptom of obstetrician’s hand
- In chronic kidney disease the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI) clinical practice guidelines advise maintaining serum levels of calcium, corrected for albumin level, within the normal range of the laboratory. Since albumin measurement is not standardized the use of albumin correction for calcium is likely to induce additional errors in bone mineral interpretation. Thus, in one study 32.6% errors of interpretation were registered.
The clinical symptoms of hypocalcemia depend on the speed of its appearance, the extent of decline, and its duration.
Symptoms depending on the calcium concentration:
- If there is a decline of iCa below 1.0 mmol/L, slight neurological symptoms such as circumoral and acral (fingers, tongue) paresthesias will occur
- If the concentration is below 0.60 mmol/L, cardiac arrest is likely
- For patients with low blood pressure or low cardiac output and iCa levels of 0.8–0.9 mmol/L a calcium treatment is required .
Especially if iCa is to be determined, the patient needs to be relaxed for at least 10 min. and should also be in a supine or sitting position for at least 5 min. prior to blood collection. The interval since the last food intake should be 4 hours. To determine iCa the blood must be collected anaerobically and tube must be completely filled. When the patient transitions from sitting to supine position, the serum total calcium after half an hour is 4.7% lower than in the sitting position . The iCa shows no change due to the change in position.
The veins should be compressed for a short period of time only with little pressure since compression for several minutes may cause a rise of total calcium by up to 10%, especially if combined with opening and closing the fist . As a result of this kind of action, there is also an increase in iCa due to acidosis.
Total calcium: serum or heparin plasma, no EDTA or citrate plasma.
Ionized calcium: lithium-heparinized whole blood, drawn in syringes or evacuated sample tubes with separator gel, are the best sample materials, if calcium-titrated heparin (40 IU heparin as the end concentration/mL of blood) is used /, /. Titration is needed because heparin binds iCa in proportion to its concentration, possibly unacceptably reducing measured concentration of iCa. One disadvantage of whole blood is that hemolysis or micro thrombi are not detected. Hemolysis above 3 g Hb/L causes a clear change in measured iCa .
Serum, anaerobically collected in evacuated tubes, is the most stable specimen for iCa. However, a partly filled evacuated tube will cause pH and subsequent iCa changes due to the loss of CO2 from the specimen .
Influence factors of total calcium
Respiratory alkalosis: in the case of hyperventilation, for rise in pH by 0.1 unit, the iCa declines by 0.2 mg/dL (0.05 mmol/L).
Exsiccosis: for each increase in albumin of 1 g/dL above the upper reference interval value, 0.8 mg/dL (0.2 mmol/L) can be subtracted from the calcium level.
Influence factors of ionized calcium
Leflunomide can interfere with ionized calcium measurement resulting in falsely decreased values most likely related to the specific structure of the ionized calcium electrode . A similar effect is seen with perchlorate, a thyroid blocking agent .
Calcium: storage at 9 °C for up to 1 week.
- Centrifuged but not separated from the erythrocytes up to 24 hours at room temperature
- Separated from the erythrocytes with the use of a separator gel up to 48 hours in an anaerobic state.
Assessment of the calcium status, if serum calcium is:
- Increased or decreased
- Normal, but clinical symptoms are present, such as bone pain, urolithiasis, renal failure, chronic diarrhea and steatorrhea or prolonged cortisone therapy.
- Differentiation between familial hypocalciuric hypercalcemia and primary hyperparathyroidism.
Atomic absorption spectroscopy (AAS)
Initially sodium is determined in the urine specimen. The calcium is then measured using a compensation solution that contains the same sodium concentration as the urine.
Urine collection over a 24-hour period (acidification in the laboratory to pH < 6.5):
- By adding 10 mL of concentrated HCl or
- By adding 1 volume of 5% glacial acetic acid to 9 volumes of urine. More recent examinations only recommend neutral collection. During the collection, the urine should be stored at temperatures of approximately 20 °C.
- 2-hour urine collection without additives collected by the patient in a fasting state between 8–10 a.m. or first morning urine for determining the calcium/creatinine ratio.
In healthy people with a normal calcium balance, the kidneys excrete the intestinally absorbed calcium. This is 6–7% of the daily dietary calcium load. If the calcium load of the diet is more than 1.5 g/day, however, hypercalciuria may occur.
- Increased intestinal calcium absorption. In this type of hypercalciuria, dietary restrictions of the calcium intake do not result in the normalization of urinary calcium excretion.
- Increased resorption of calcium from the skeleton, even on a low-calcium diet (resorptive hypercalciuria). This type of hypercalciuria is not associated with a secondary hyperparathyroidism. The patients on a low-calcium diet have a negative calcium balance resulting in significant bone resorption and an increased risk of fracture.
- Reduced renal-tubular calcium reabsorption. This type should be authoritative for a maximum of 5% of the hypercalciurias.
PH or idiopathic hypercalciuria is a form of Ca excretion, with which on a diet of 1.0 to 1.2 g of calcium and 1.0 to 1.5 g protein/kg of body weight per day there is increased Ca excretion. Most of these patients have absorptive hypercalciuria due to increased enteral calcium absorption, with resorptive hypercalcemia being the second most common cause. The latter is connected to a certain extent with demineralization of the skeleton, but the most frequent association is with calcium nephro lithiasis, thus, the incidence of hypercalciuria for kidney stone patients is 50–60%.
In addition to racial, geographic and seasonal fluctuations, gender and weight, nutrition also influences calcium excretion. Important influence factors are the contents of sodium, potassium, phosphorus, carbohydrates and protein in food, and alcohol consumption. When the intake of sodium is increased, the calcium excretion in the urine increases by 20–40 mg/day for each 2.3 g of sodium intake (corresponding to 5.8 g of table salt) .
Due to increased renal synthesis of 1,25 (OH)2D hypophosphatemia also leads to increased intestinal calcium absorption. An increased supply of protein also increases calcium excretion. A moderate supply of protein (1–1.5 g/kg of body weight per day) does not change the calcium metabolism. The causes of secondary hypercalciurias are listed in .
The following procedures are carried out to confirm an increased calcium excretion:
- Assay of the calcium/creatinine ratio in the morning urine. A ratio ≥ 0.57 is indicative of hypercalciuria in adults
- Investigation of one to two 24-hour collected urine samples. An excretion of ≥ 300 mg in men and ≥ 250 mg in women indicates hypercalciuria.
The patient should not alter his dietary habits 24 hours before and during the collection of urine samples.
A calcium excretion in the reference interval of the 24-hour collected urine does not rule out relative hypercalciuria because the calcium excretion can be too high relative to the calcium intake with the diet ().
When clarifying hypercalciuria, a differentiation must be made between primary and secondary causes, with the latter including primary hyperparathyroidism.
If hypercalciuria has been diagnosed and secondary causes of the increased calcium excretion have been ruled out, it is necessary to differentiate metabolic causes from primary hypercalciuria . To do this, the calcium/creatinine ratio is determined with a restricted diet of calcium, protein and sodium and evaluated as shown in :
- After 1 week on the diet (1.0 to 1.2 g of protein per kg of body weight, calcium ≤ 400 mg/day, sodium 100 to 150 mmol/day, corresponding to 5.8– 8.7 g table salt/day) and fasting overnight, the calcium/creatinine ratio is determined in the spontaneous morning urine.
- If the calcium/creatinine ratio falls below 0.57 too much calcium is enterally absorbed with a regular diet. Absorptive hypercalciuria is the result.
- If the calcium/creatinine ratio does not normalize (ratio ≥ 0.57) despite calcium restriction, the result is fasting hypercalciuria. There is increased calcium resorption from the bone (resorptive hypercalciuria). If PTH is also determined, its increase indicates renal hypercalciuria.
The calcium excretion must be evaluated relative to the amount of dietary calcium. For example, if the calcium excretion for a daily intake of 800 mg (20 mmol) of calcium is less than 50 mg (1.3 mmol) in 24 hours, the result is hypocalciuria. Such a finding is typical for osteomalacia related to a vitamin D deficiency.
In cases of hypercalcemia or calcium levels near the upper reference interval value and increased PTH concentration, the calcium excretion in the 24-hour urine must be determined to differentiate the familial hypocalciuric hypercalcemia (FHH) from primary hyperparathyroidism. Calcium excretion below 100 mg (2.5 mmol/L)/24 hours or a urine calcium/creatinine ratio (mmol/mmol) below 0.01 are indicative of FHH.
Method of determination
Accurate results are obtained by means of atomic absorption spectrometry. Results determined by flame photometry are only accurate if a compensation solution is used with a sodium concentration identical to that of the urine to be examined.
Urinary calcium excretion
Under alkaline conditions, the divalent cations calcium and magnesium have the tendency to precipitate in the urine, primarily as phosphates such as brushite (CaHPO4 × 2 H2O) and struvite (MgNH4PO4 × 6 H2O). As a result, magnesium, calcium and phosphate in the urine are determined to be too low. Therefore, the urine should be acidified (pH under 6.5) in the laboratory before the measurement. One recent examinations shows no difference between acidified and neutral urine.
Approx. 1% of total body calcium is renewed daily. On a systemic level, a complex interaction between PTH, 1,25(OH)2D and calcium allows the organism to maintain the calcium homeostasis, despite variations in the daily intake.
PTH has a regulating effect as follows:
- Stimulation of the kidneys for the reabsorption of calcium
- Release of calcium from the skeletal system
- Enteral absorption of calcium.
By activating the enzyme 25-OHD-1α-hydroxylase in the kidneys, PTH stimulates the synthesis of 1,25(OH)2D (calcitriol) from 25(OH)D.
Calcitriol has the following effects:
- Increase of the Ca absorption in the duodenum and upper jejunum via calcium-binding proteins. The increase in the Ca concentration in the blood, on the other hand, suppresses PTH production.
- Control of the PTH concentration by suppressing the genetic transcription of pre-pro-PTH.
Ca is excreted via the kidneys and the intestines. Glomerularly filtrated Ca is reabsorbed up to 94–96% (tubular). The Ca amount in urine is up to 300 mg (7.5 mmol)/24 hrs. Ca excreted together with the digestive juices in the intestines is reabsorbed up to 90% ().
The G protein-linked calcium-sensing receptor (CaSR), a guanine nucleotide-binding protein (G protein), is the main mediator on the cellular level of the calcium homeostasis. It is expressed on the cell membrane of the parathyroids and the renal tubular cells. Ca directly activates the CaSR, suppresses the PTH excretion, and reduces the renal-tubular reabsorption of Ca. The activation of the CaSR in hypercalcemia causes the G protein-mediated stimulation of the phospholipase C by the proteins Gq and G11. The result is an increase of inositol 1,4,5-triphosphate and an accumulation of intracellular calcium. These changes lead to a decrease in the PTH level and an increase in the renal excretion of calcium.. Thus, a high concentration of calcium suppresses the vasopressin-induced water reabsorption in the collecting ducts via the CaSR and thus declines the capacity of urine concentration. This is the case, for example, with hypercalcemias over 12 mg/dL (3.0 mmol/L), which cause hypercalcemic nephrogenic diabetes.
Parathyroids of patients with terminal kidney failure have a reduced number of CaSR. Therefore, extracellular Ca is only insufficiently perceived, with the result of secondary hyperparathyroidism and its systemic effects. It is treated with the allosteric modifier of the CaSR, the cinacalcet, which causes activation of the CaSR. Its use leads to a decrease in the elevated PTH concentration and in the Ca × P product in dialysis patients.
Mutations in the gene that encodes the CaSR lead to a suppressing or activating effect with hypercalcemic or hypocalcemic disorders.
- The parathyroids are resistant to calcium and only respond to an increased calcium concentration with suppression of the PTH excretion. The cause is that the extracellular domain of CaSR is changed by mis- sense mutations in such a way that a reduced number of functional receptors is expressed on the cell membrane.
- There is an increased reabsorption of calcium in the thick ascending limb of the nephron.
Hypocalcemias are caused by absent or hypofunctioning parathyroids, vitamin D deficiency due to reduced enteral absorption, UV light deficiency, or by hepatic or renal defects in the metabolism of vitamin D.
In chronic renal insufficiency with decreased phosphate clearance, the diminished synthesis of 1,25(OH)2D and the existing hyperphosphatemia with subsequent hypocalcemia are the causes for the development of secondary hyperpathyroidism (sHPT) and of hyperactive bone disease or so-called renal osteopathy (osteomalacia, osteodystrophia fibrosa) . The renal insufficiency associated decline in 1,25(OH)2D diminishes gastrointestinal absorption of Ca; however, passive diffusion of Ca2+ continues and may lead to a positive Ca balance, aggravated by diminished urinary Ca excretion due to sHPT. Increased Ca release from bone in hyperactive renal bone disease enhances the positive Ca balance and may worsen vascular calcification . Hyperactive bone disease leads to an increased Ca resorption from the bone because of increased secretion of PTH (secondary hyperparathyroidism).
Hypercalcemia is based on increased mobilization of calcium from the bone, increased intestinal absorption and/or increased renal tubular reabsorption.
In the case of pHPT, a singular adenoma or hyperplasia of all parathyroids is the cause of autonomous PTH excretion and of hypercalcemia. The result can be the formation of kidney stones, renal calcification, osteodystrophia fibrosa, pancreatitis, and duodenal ulcers. Metastatic calcifications are mainly observed with an increase of the calcium phosphate product to values above 60–75 (calculation based on values in mg/dL) .
Hypercalcemia is a frequent complication of malignant tumors in the advanced stage. Two mechanisms are essentially responsible for tumor-associated hypercalcemia:
- Metastatic tumor cells cause osteolysis by direct contact with the bone. Factors with a local effect such as prostaglandin E2, interleukin-1 as well as tumor necrosis factors are said to be released. This mechanism is common in cases of multiple myeloma and breast carcinoma.
- Humoral factors, which lead to an effect like PTH (i.e., increased Ca mobilization from the bone, increase of the cyclic AMP and phosphaturia). However, unlike pHPT, there is no increase in 1,25(OH)2D, instead, it is rather decreased. One such humoral factor which is formed by the tumor and bound to the PTH receptors is the parathormone-related peptide (PTHrP). In cases of PTHrP forming metastases, circulus vitiosus develops. During this, PTHrP releases growth factors such as TGF-β from the bone matrix, which, in turn, increase the PTHrP excretion of the tumor tissue .
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The terms phosphate and phosphorus are used interchangeably in laboratory medicine. This is meaningless for clinical purposes, because the phosphate content of a sample is specified as elementary, inorganic phosphorus (Pi). The Pi is present in serum in the form of orthophosphate which is derived from the sequential ionization of phosphoric acid (). Pi occurs only in the organism as H2PO4– and HPO42– , because neither H3PO4 as a strong acid nor PO43– as a strong base is compatible with a physiologic pH. At a pH of 7.4, the molar ratio of HPO42–/H2PO4– is 4 : 1. It decreases in acidosis and increases in alkalosis . It is customary for clinical laboratories to report phosphate concentration in terms of Pi. Thus a 1 mmol/L solution of phosphorus (atomic weight 31) is equivalent to a serum level of 3.1 mg/dL.
At neutral pH, the 4 : 1 molar ratio carries a total of nine negative charges, making the every charge per mmol –9/5 or –1.8. Thus, a 1 mmol/L solution of orthophosphate at pH 7.4 is equivalent to a 1.8 mEq/L solution . Consequently, in serum, the following measurement units for Pi are equal to each other: 1 mmol/L = 3.1 mg/dL = 1 mEq/L .
The determination of Pi in serum is often not sufficient for the assessment of the phosphate status, thus necessitating the determination of urinary phosphate.
- Bone disease
- Chronic kidney disease, dialysis patient
- Status post thyroid operation
- Parathyroid disease
- Kidney stone patient
- Chronic alcoholism
- Intensive care medicine (parenteral nutrition, mechanical ventilation)
- Cardiovascular disease
- Suspicion of vitamin D deficiency (malabsorption)
- Muscle weakness, bone pain.
Principle: reaction of Pi with ammonium molybdate to form a phospho molybdate complex. The colorless complex is measured after reduction by reducing reagents to produce molybdenum blue which is measured spectrophotometrically at 580 nm.
Serum, heparin plasma (sample taken in fasting state in the morning before 10 a.m. based on circadian rhythms, see Comments and problems): 1 mL
Approximately 85% of phosphorus is localized in the bones in connection with calcium and 14% is intracellular. Most intracellular phosphorus is organically bound to carbohydrate intermediates, lipids, and proteins, and a smaller fraction is inorganic . Phosphate is the main intracellular anion and its metabolism is closely linked with that of calcium . The daily phosphorus intake in diet is approximately 1 g (32 mmol). A low protein diet decreases phosphorus intake. However the quantity and bio availability of phosphorus differ according to the type of protein. The gastrointestinal absorption of phosphorus, mostly in the form of phytate, is lower from plants (along with fibers) than from meat (30 to 50% vs. 50 to 70%). Restricting dietary phosphorus intake to less than 800 mg (26 mmol) per day is recommended for moderate-to-advanced kidney disease .
Although only 1% of the phosphorus content of the organism is present in plasma and other bodily fluids, the serum concentration correlates to the body content in most cases. Renal reabsorption of phosphate is the most important factor of the phosphorus level in serum. If the phosphorus reabsorption increases due to an elevated intake or a decrease in glomerular filtration rate, the renal reabsorption of phosphate is reduced. The renal reabsorption of phosphorus is regulated by the fibroblast growth factor 23 (FGF-23) and the parathyroid hormone. The concentration of both hormones increases with a rise in the serum phosphorus concentration. The renal reabsorption of phosphorus can be determined by measuring the TmP/GFR (see ).
- Redistribution disturbance between the intracellular and extracellular compartment
- Decreased supply or reduced intestinal absorption of phosphorus
- Renal loss of phosphorus because of insufficient vitamin D effect.
In the intracellular space, phosphorus is primarily involved in the carbohydrate and lipid metabolism or is bound to proteins. Only a small proportion is inorganic. Conditions that favor organic cellular phosphorus binding result in a shift of phosphorus from the extracellular to the intracellular space, thus lowering serum phosphorus concentration.
- Glucose-induced insulin release that promotes the transport of glucose and phosphate ions into liver and muscle cells
- Administration of fructose stimulates phosphorylation phosphate ions are metabolized in the intracellular compartment and replaced from outside the cell
- Metabolic acidosis, respiratory alkalosis, recovery from hyperparathyroidism, and physiologic concentrations of catecholamines may cause mild and transient phosphorus shift to the intracellular compartment. The maldistribution may become severe if phosphate stores are depleted because of disease or malnutrition .
- Extended fasting
- Fluid losses owing to surgical trauma
- Rapidly proliferating tumors, which metabolize a lot of phosphate
- Acute-phase reaction (e.g., sepsis, postoperative state)
- Nonabsorbable antacids (e.g., aluminium hydroxide) which bind intestinal phosphate and limit the gastrointestinal absorption.
The external supply of phosphorus is reflected by the urinary phosphate ion (P) excretion (UPV). The UPV is the product of the urinary P concentration (UP) in mg/dL and the urine volume (V). The UPV corresponds to the enteral absorption of phosphorus. In cases of malnutrition (alcoholism), malabsorption, in intensive care patients and in treatment with phosphate binders, enteral phosphorus uptake and UPV are reduced. The UPV is then below 600 mg (20 mmol)/24 h.
With normal glomerular filtration rate (GFR), the renal threshold of phosphate (i.e., UpV/GFR) is the dominating factor for the regulation of the phosphate status. With a decrease of GFR, the UpV increasingly gains importance in its influence on the serum concentration of phosphorus. Tubular phosphate reabsorption usually ranges from 80–90% and is essentially influenced by PTH, vitamin D, calcitonin, antidiuretic hormone, growth hormone, thyroid hormones, estrogens and glucocorticoids.
The renal excretion of phosphate is dependent on the (TmP/GFR) which is dependent on the renal transport maximum of phosphate (TmP) and the glomerular filtration rate (GFR) /, /. TmP, also known as phosphate threshold is the proportion (%) of phosphate in primary urine which is reabsorbed in the proximal renal tubules (more than 80% in healthy individual). An increased TmP/GFR (mmol/L) can be found in acquired and hereditary bone mineralization disorders.
- Hypophosphatemia due to increased renal phosphate clearance
- An inadequate low value of 1,25(OH)2D
- A variable bone disease (osteomalacia, rickets, renal bone mineralization disease).
Mild hypophosphatemia is relatively common in hospitalized patients and is reported in up to 30% of the surgical cases . Clinically relevant hypophosphatemia below 1.5 mg/dL (0.48 mmol/L) and serious forms with a level below 1 mg/dL (0.32 mmol/L) are rare. The prevalence of the serious form in hospitalized patients is 1–2 per thousand . The symptoms of serious hypophosphatemia are muscular weakness, muscle pain, central nervous system symptoms such as confusion, convulsions, and coma. Muscular weakness can lead to respiratory failure. Hematological disorders such as hemolytic anemia and a dysfunction of the neutrophil granulocyte can also occur.
For causes of hypophosphatemia refer to:
Hyperphosphatemia reduces the concentration of 1,25 (OH)2D and increases the levels of PTH and fibroblast growth factor 23 (FGF23). Both hormones promote urinary phosphate excretion.
Elevated levels of PTH and FGF23 can cause renal bone disease, left ventricular hypertrophy, vascular calcification, and accelerated progression of kidney disease from vascular and tubulointerstitial injury .
The effect of serum phosphorus on kidney disease itself remains uncertain. In non dialysis chronic kidney disease and non chronic kidney disease populations, small increases in phosphorus levels, even within normal ranges, have been shown to be associated with greater mortality and cardiovascular outcomes. In subjects with advanced chronic kidney disease, higher serum phosphorus levels have been shown to predict increased incidence of end-stage renal disease, suggesting that phosphorus elevations may adversely affect kidney function .
- Disturbance of phosphate homeostasis between the intracellular and extracellular compartment
- Acute and chronic renal failure
- Increased renal phosphate reabsorption (hypoparathyroidism)
- Increased dietary intake of phosphates, increased intestinal phosphate absorption or intravenous phosphate supply (e.g., during treatment with the antibiotic fosfomycin).
Disturbance of phosphate homeostasis between the compartments
An increased phosphate shift from the intracellular to the extracellular compartment occurs in cases of:
- Acidosis such as respiratory acidosis, tissue ischemia, lactic acidosis, and diabetic ketoacidosis
- Tissue trauma (rhabdomyolysis, hemolysis, cytostatic treatment, malignant pyrexia).
The renal insufficiency associated decline in 1,25 (OH)2 and reduction of GFR in chronic kidney disease diminishes urinary phosphate excretion resulting in hyperphosphatemia. This causes hyperphosphatemia, a condition that results in the development of secondary hyperparathyroidism and mineral renal bone disease .
The capacity of the kidneys for excreting phosphate is high with normal GFR. Overt hyperphosphatemia is infrequent in stages 1, 2, and 3 of chronic kidney disease (CKD), however in stages 4 and 5 the GFR is below 30 [mL × min–1 × (1.73 m2)–1] and overt hyperphosphatemia is frequent. Secondary hyperparathyroidism develops, caused by phosphate retention and reduced renal 1,25(OH)2D (calcitriol) synthesis. Calcitriol stimulates the intestinal calcium absorption and suppresses the secretion of PTH. Impaired renal production of calcitriol, the active form of vitamin D, contributes to the generation and maintenance of secondary hyperparathyroidism. Calcitrol represses parathyroid cell proliferation and PTH synthesis. Calcitriol deficiency results in direct secondary hyperparathyroidism due to decrease in intestinal absorption of calcium.
The Kidney Disease Outcomes Quality Initiative (NKF/KDOQ1) Clinical Practice Guidelines for Bone Metabolism and Disease recommend that the phosphate levels in the serum should be kept constant:
- In stage 3–4 of CKD, in the range of 2.7–4.6 mg/dL (0.87–1.49 mmol/L)
- In the range of 3.5–5.5 mg/dL (1.13–1.78 mmol/L) for dialysis patients.
High levels of PTH and phosphate result in systemic disorders such as cardiovascular disease (CVD) and increased mortality in patients with CVD. In chronic dialysis patients with phosphate levels in serum above 6.5 mg/dL (2.1 mmol/L) and a product Ca × phosphate above 72 mg2/dL2 the prevalence of CVD is 40% and of left ventricular hypertrophy 70%. Cardiovascular mortality is 10–20 times higher than in the general population of the same age.
- A decrease in calcium and 1,25(OH)2D concentration and a reduction of intestinal calcium absorption. For tumor calcinosis, pseudoxanthoma elasticum, cortical hyperostosis and thyrotoxicosis, there is no decrease of calcium.
- Ectopic calcifications in various organs and vessels, particularly in end stage renal failure. The risk of calcifications begins with a Pi serum level above 5.5 mg/dL (1.8 mmol/L) and a product of Ca (mg/dL) × phosphate (mg/dL) above 60–75 with the presence of an alkaline pH. Calcium phosphates precipitate in the form of hydroxyl apatite crystals in the tissues.
- Whether phosphate is added to the extracellular compartment from a variety of exogenous or endogenous sources or
- Whether the urinary excretion of phosphate is reduced from either decreased GFR or increased tubular reabsorption.
Severe hyperphosphatemia with a concentration ≥ 14 mg/dL (4.5 mmol/L) is always invariably multi factorial, usually resulting from addition of phosphate to the extracellular compartment together with decreased phosphate excretion .
The determination of urinary phosphate excretion by itself is often insufficient for assessing the phosphate status, because the excretion is dependent on dietary intake, bone metabolism, GFR, and renal phosphate reabsorption. Therefore, clearance methods are used for assessing the phosphate excretion. The following examinations are conducted depending on the clinical concerns:
- Phosphate clearance
- Percentage of tubular phosphate reabsorption
- Tubular maximum for phosphate reabsorption.
Suspected tubular syndromes associated with phosphate losses.
Primary and secondary parathyroid dysfunctions.
Principle: the object is to determine the plasma volume that is cleared of phosphate per minute. The phosphate clearance test is carried out in two 1-hour collection periods according to the following test protocol:
- 7.00 a.m., the fasting patient drinks 500 ml of tea
- 8.00 a.m., the patient empties the bladder in the toilette bowl and then drinks another 250 mL of tea
- 9.00 a.m., the patient empties the bladder in the first container, blood is collected for the determination of phosphate.
- 10.00 a.m., the patient empties the bladder in the second container.
Determination of phosphate (P) in serum and in both urine collection specimens, measurement of the urinary excretion over the course of both collection periods.
Calculation of phosphate clearance (Cp)
The Cp of both collection periods is determined.
The excretion of phosphate in the 24-hour urine in individuals with a normal phosphate supply is 0.6–1.55 g (20–50 mmol).
The Cp has an advantage over the determination of the phosphate excretion that the phosphate excretion is assessed relative to the serum phosphate level. Physiological increases of Cp are measured in increased alimentary supply of phosphate and NaCl and physiological decreases in growth spurt, gravidity and the lactation period. Diseases with increase in Cp are shown in .
The Cp does not take renal function into consideration. Thus, for primary hyperparathyroidism with limited renal function, the Cp is often normal.
For the differential diagnostic assessment of pathological Cp values the following further examinations are important:
- Calcium, phosphate, serum protein, chloride, creatinine, and alkaline phosphatase in serum.
- Estimated glomerular filtration rate and calcium excretion in urine.
Detection of renal tubular phosphate reabsorption defect:
- Primary and secondary disorders of the parathyroid function
- Tubular syndromes with phosphate leak.
Principle: the percentage of renal tubular phosphate reabsorption (TRP%) is determined as follows:
- 2-hour urine collection in the morning after the bladder has been emptied. The patient should be in a fasting state. Measurement of urine volume, creatinine and phosphate.
- Blood collection at the midpoint of the collection period for determination of creatinine and phosphate
- Calculation of the tubular reabsorption of phosphate according to the following formula:
Cp = Phosphate clearance, Ccr = Creatinine clearance
The TRP (%) considers, in contrast to the Cp the renal function. It is used for differentiating hypophosphatemia. One disadvantage is the dependency on the supply of phosphate. Thus, if phosphate is withdrawn, a healthy individual can have a TRP (%) above 90%, compared to below 82% with a high supply of phosphate. The evaluation of the (TRP%) for diseases is shown in .
The TmP/GFR, also called renal phosphate threshold, describes the maximum phosphate concentration (TmP) in the glomerular filtrate, below which all of the filtered phosphate is tubularly reabsorbed. It is possible to determine the TmP/GFR directly, but is technically very demanding. Therefore, the renal phosphate threshold is generally determined according to the nomogram of Walton and Bijvoet . Refer to .
Detection of a renal tubular phosphate reabsorption defect.
Principle: the percentage of renal phosphate reabsorption is determined.
- 2-hour urine collection in the morning after the bladder has been emptied. The patient should be in a fasting state. Measurement of urine volume, creatinine and phosphate.
- Blood collection at the midpoint of the collection period for determination of creatinine and phosphate
- Calculation of the tubular reabsorption of phosphate according to the following formula:
Cp = Phosphate clearance, Ccr = Creatinine clearance
- The phosphate threshold (TmP/GFR) is read off the nomogram (), based on the serum phosphate concentration and the TRP value. The TmP/GFR is determined from the nomogram as follows: phosphate concentration of the serum and TRP or Cp/Ccr value are connected by using a ruler in such a manner that the right side intersects the TmP/GFR axis. This point of intersection corresponds to the phosphate threshold of the patient in mg/dL or mmol/L in the glomerular filtrate.
The TmP/GFR behaves in parallel with the phosphate concentration in serum, which means that it depends on the phosphate threshold as it changes throughout the day. Between 11 a.m. and 3 p.m., there is a simultaneous increase of phosphate in serum with the phosphate excretion in urine and the TmP/GFR .
The TmP/GFR is decreased in cases of tubular syndromes with a loss of phosphate such as phosphate diabetes and the hyperparathyroidism . A tubular phosphate threshold below 2.2 mg/dL (0.70 mmol/L) is seen in 20% of the patients who are developing kidney stones, but in only 5% of the control patients .
The phosphate content of food influences the serum phosphate concentration. To minimize this influence, the blood sample should be collected in the morning after fasting overnight. Serum phosphate shows a circadian rhythm with a nadir early in the morning, a plateau late in the afternoon, a slight decline late in the evening, and a plateau at night. The difference between the highest and lowest value is 30% and the absolute value is around 1.2 mg/dL (0.39 mmol/L) /, /.
Serum is preferred over plasma; the serum level is 0.2–0.3 mg/dL (0.06–0.10 mmol/L) higher than in plasma.
The serum must be separated from the erythrocytes within 2 hours, because phosphate escapes from the blood cells depending on the temperature, causing falsely elevated values.
Hemolytic serum is not acceptable, because phosphate is cleaved from organic phosphate esters that are released in the serum. Theoretically, the measured phosphate can be increased by up to 20%, something which is not seen in practice, however.
Thrombocytosis is also associated with increased phosphate values.
Method of determination
In addition to hemolysis, hyperlipidemia and hyperbilirubinemia interfere with the phosphate assays. Obfuscations interfere due to spectral interference, because the phospho molybdate method used in most analysis systems measures at 340 nm .
The unmodified acid ammonium molybdate method produces 19% spuriously high results. The false increase of Pi concentration is attributable to formation of precipitate in the reaction mixture. The precipitate is formed by interaction between immunoglobulins and the unmodified acid molybdate reagent. Diluting of the sample to about 40 g/L total protein reduces, but does not always eliminate the interference .
Phosphorus is one of the essential structural components of cells and organelles and it plays an active part in the generation, storage and release of metabolic energy. In particular, phosphate is involved:
- As a part of nucleoproteins, nucleic acids and in the form of phospholipids in membranes
- In the production of ATP during the oxidative phosphorylation in the mitochondria
- In the glycogenolysis and glycolysis
- In the form of 2.3 diphosphoglycerate in regulating the dissociation of oxygen from oxyhemoglobin
- In enzymatic processes the phosphorylation of enzymes is an important control mechanism of enzymatic functions. In addition, phosphate is involved in many enzymatic reactions in the form of cyclic adenosine and guanine nucleotides as well as NADP.
- In many functions the organism needs ATP such as muscle contraction, central nervous system processes, and the transport of electrolytes
- AS an essential component of bones.
All of the above-mentioned functions are impacted by a phosphate deficiency.
The concentration of intracellular phosphorus is approximately 3.1 mg/dL (1 mmol/L). Most of the intracellular phosphorus exists in the form of organic compounds, (lipids, carbohydrate intermediary products) and plays a role in transport processes, and cell growth. Conditions that promote the binding of organic phosphorus, such as the administration of insulin for hyperglycemia, lead to a shift of phosphate from the extracellular to the intracellular compartment.
Metabolic disorders that involve acidosis lead to a decrease in the oxidative phosphorylation, and the glycolysis, and hydrolysis of intracellular phosphate esters. The released phosphate is shifted to extracellular and leads to an increase in phosphate. In cases of deficient phosphate supply or catabolic metabolism, the concentration of plasma phosphate remains constant for a long time despite the renal excretion of phosphate because intracellular phosphate is continuously resupplied. Hypophosphatemia only occurs if an anabolic metabolism is restored and phosphate is shifted from the extracellular to the intracellular compartment. In cases of severe malnutrition, high amounts of infused glucose can be fatal due to severe hypophosphatemia that can develop.
Phosphate is present in many foodstuffs such as meat and vegetables. The mean daily intake of dietary phosphate is 1,000 mg of which approximately 70% is absorbed in the small intestine. Approximately 200 mg of phosphate derived from endogenous sources is secreted into the intestines daily and are reabsorbed.
The tissue of a male person weighing 70 kg contains approximately 0.7 kg of phosphate, of which 85% is located in the skeleton, approximately 15% is contained in the soft tissues, and 0.3% is in the extracellular compartment. The bone is the body’s store for phosphate and the kidneys are the regulator of the phosphate homeostasis. With a phosphate plasma level of 3.4 mg/dL (1.1 mmol/L) and a GFR of 180 liters/24 hours, 6.3 kg of phosphate is filtered. Approximately 80% of the filtered load is reabsorbed in the proximal and 10% in the distal tubules. The capacity of the kidneys to reabsorb phosphate is determined by the tubular maximum (Tm) per mL of GFR (Tm/GFR). The kidneys regulate the excretion of phosphate depending on the intake. Reduced intestinal phosphate intake is responded to by an elevation of the TmP/GFR, increased intestinal phosphate intake by a decrease.
With chronic renal failure, hemodynamic and morphological changes occur in order to maintain the phosphate homeostasis . These include hypertrophy of nephron segments and the tubular phosphate handling. The TmP/GFR of the remaining nephrons is increased, which allows the phosphate plasma concentration to be kept normal for a long time.
The homeostasis of phosphate and thus the phosphate serum concentration is maintained by mechanisms which regulate the intestinal absorption of phosphate. If the concentration of plasma phosphate is increased due to increased intake with the diet or due to the decrease in GFR, the enteral phosphate resorption decreases. In this situation, the regulation is done via the increase of FGF23, which reduces the reabsorption of phosphate in the kidneys and causes phosphaturia. PTH has the same effect.
Between 60–80% of the phosphate is resorbed in the small intestine and the net excretion takes place via glomerular filtration minus the tubular reabsorption. Intestinal absorption and renal reabsorption occur via sodium-dependent co transporters ().
The kidney tubular brush border membrane contains the co transporters NPT2a and NPT2c. The small intestine primarily contains NPT2b. NTP2a transports 3 sodium ions with 1 phosphate anion and NTP2c only 2 sodium ions with 1 phosphate ion. The expression of NPT2a and NPT2c in the brush border membrane of the kidneys is rapidly down regulated by an increase of phosphate, FGF-23 and PTH, but NPT2c of the small intestine only does this after several days . Inactivity of the genes of the renal co transporters induces hypophosphatemia through increased excretion of phosphate. The consequences are demineralization of the bone and the formation of kidney stones.
The phosphate level in serum is approximately 4 mmol/L, of which 70% is present in organic form, primarily in the form of phospholipids. The remainder is inorganic phosphate, of which 70% is in free form, 15% in protein-bound form, and 5% is present bound to magnesium or calcium.
Serum phosphate level is regulated by intestinal phosphate absorption, renal phosphate handling and equilibrium of extracellular phosphate with that in bone or intracellular fluid. PTH, 1,25(OH)2D and FGF 23 regulate serum phosphate by modulating intestinal phosphate absorption, renal phosphate reabsorption and/or bone metabolism.
The effects of PTH on the kidneys are as follows: PTH binds to the PTH receptor 1 of the proximal tubular cells, stimulates the synthesis of cyclic AMP and phospholipase C, and reduces the renal phosphate reabsorption by preventing the expression of NPT2a (). PTH in addition increases the renal phosphate excretion, but it is unknown whether PTH has a direct effect, because changes of the phosphate concentration always involve changes of calcium. It is assumed that PTH has no direct phosphaturic effect .
The FGF-23 protein consists of 251 amino acids, and is formed in osteocytes and also in other tissues during the developmental phase of the organism. It exerts its effect via FGF receptors (FGFRs), which are bound to the transmembrane protein klotho (). Klotho is a co receptor, which increases the sensitivity of the FGFRs for FGF23. FGF23 acts directly on the parathyroid to decrease serum PTH. In absence of functioning klotho, plasma concentration of FGF23 is high, but ineffective in decreasing the PTH level.
FGF23 induces phosphaturia by decreasing phosphate reabsorption in the proximal tubule and inhibits renal 25-OHD-1α-hydroxylase leading to decreased conversion of 25(OH)D to 1,25 (OH)2D. Excess of FGF 23 causes marked hypophosphatemia, renal phosphate wasting, and an inappropriate low 1,25 (OH)2D level for the degree of hypophosphatemia . The plasma FGF23 concentration increases with a decline in the GFR and correlates to the phosphate concentration in serum and the fractional secretion of phosphate serves to differentiate patients with volume depletion who respond to saline intake with correction of the alkalosis from those who do not respond. The early increase of FGF23 in CKD prevents hyperphosphatemia through increased renal phosphate excretion and decreased intestinal absorption of phosphate. By suppressing the 25-OHD-1α-hydroxylase and its associated decrease of 1,25 (OH)2D increased levels of FGF23 can cause secondary hyperparathyroidism (sHPT). The relationships between calcium, phosphate, PTH, FGF23 and 1,25(OH)2D are shown in .
Dialysis patients have significantly increased FGF23 values and the elevation indicates the development of refractory sHPT. The restriction of phosphate leads to a decline of FGF23, a decreased renal phosphate reabsorption and, due to increase of 1,25 (OH)2D the intestinal phosphate absorption is enhanced .
Approximately 90% of patients with stage 3 and 4 of chronic kidney disease have increased concentrations of FGF23 without hyperphosphatemia and a study over 53 months has shown that patients with higher FGF23 values have a greater progrediens in renal insufficiency than those with lower values . Increased FGF23 levels at the start of dialysis are also associated with higher mortality in the first year of dialysis. According to a study patients in the highest of 4 quartiles had a 6-times greater risk of mortality than those in the first quartile of the FGF23 values.
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PTH works as an interface between bone , calcium metabolism and vitamin D-metabolism. PTH regulates normal bone and mineral ion homeostasis and is central in the pathogenesis of bone disease, in primary and secondary hyperparathyroidism, and especially in advanced renal insufficiency .
PTH is a 84-amino acid polypeptide [PTH (1–84)] produced by the parathyroid glands and its partial degradation within these glands results in the secretion of intact hormone and various fragments into the peripheral blood. It exerts its classical biological effects on bone and kidney through its first 34 amino acids, that is, its N-terminal structure . The PTH activity is mediated via interaction with the PTH receptor PTH1.
PTH exerts the calcium concentration in plasma through:
- Release of calcium from the bones
- Increase of the renal-tubular calcium reabsorption.
- Stimulation of the renal 1,25(OH)2D (calcitriol) formation which increases the calcium absorption in the small intestine. The resulting increase of calcium in peripheral blood inhibits the excretion of PTH via feedback inhibition.
- Phosphaturic effect due to suppression of the sodium-phosphate co transporter in the proximal tubular cells of the kidney.
In addition to the intact PTH (1–84), PTH fragments formed in the parathyroids or produced by the liver circulate in plasma. Following release PTH (1–84) undergoes proteolytic degradation in Kupffer cells in the liver. The resulting carboxy-terminal (C-terminal) PTH fragments but not the N-terminal fragments re-enter the circulation. The PTH fragments and intact PTH are excreted renally. C-terminal fragments have a 5–10 times longer half-life than the intact PTH. As a consequence of the different half-lives, the following proportions of immunoreactive PTH are measured in normocalcemic patients:
- Approximately 5–30% intact PTH (1–84)
- Approximately 70–95% C-terminal fragments
- Approximately 4–8% N-terminal fragments.
- Aminoterminal structure of 34 amino acids which exerts its biological effects through its first 34 amino acids (N-terminus). It binds to the type 1 PTH/PTHrP receptor and activates the Gs protein-mediated signal transmission in the target organs (see ). The first 6 amino acids of the N terminus are important for binding to the type 1 PTH/PTHrP receptor. Most immunoassays for the determination of PTH 1–84 (intact PTH assays) have their main epitope in region 13–34 of the PTH (1–34) structure, early epitopes like 12–18 and 13–24 being more frequent than distal epitopes 26–32 .
- Carboxy-terminal structure of the last 50 amino acids, which has no direct influence on the type 1 PTH/PTHrP receptor.
C-PTH fragments: these fragments lack a smaller or larger part of the N terminus. In healthy individuals the proportion is approximately 80% and in chronic kidney disease as much as 95%. C-PTH fragments are acutely regulated in the circulation by ionized calcium. This regulation operates within the reference interval of calcium. Hypocalcemia favors the secretion of PTH (1–84) over the C-PTH fragments .
N-PTH fragments: such fragments are no proteolytic degradation products of PTH (1–84) in the liver, but are secreted by the parathyroids and make up 4–8% of the immunoreactive PTH. In the final stage of chronic kidney disease, the proportion can increase to 15%. N-PTH fragments are also increasingly secreted in patients with primary hyperparathyroidism.
Non-(1–84) (PTH) fragments
The non-(1–84) PTH fragments or amino-terminal (N)- truncated PTH fragments are large circulating carboxy-terminal C-fragments with a partially preserved N structure. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1–84) PTH fragments. The non-(1–84) PTH fragments differ from other circulating C-PTH fragments by their capacity to react with the 13–34 structure in intact PTH assays . Their proportion of the C-terminal fragments is approximately 10% and, in healthy individuals, approximately 20% of the PTH measured with intact PTH assays. In chronic kidney disease, the proportion of non-(1–84) PTH fragments can be up to 45%.
One important non-(1–84) PTH fragment is PTH 7–84. It does not bind to PTH/PTHrP 1, is biologically inactive, and has no calcium regulating effect. However, PTH 7–84 binds to receptors, which react with the C-terminal part of PTH (C-PTH receptors). These receptors are located on osteolytic cells. PTH 7–84 suppresses bone resorption, which is triggered by osteoclast-activating substances such as 1,25(OH)2D, prostaglandin E2 and IL-11. PTH 7–84 is also supposed to have hypocalcemic properties and counteract the calcium increasing effects of PTH.
- Differentiation between hyperparathyroidism-related hypercalcemia and other forms of hypercalcemia
- Assessing the bone metabolism and therapeutic monitoring in patients with chronic kidney disease
- In patients with vitamin D deficiency
- In patients malabsorption syndrome
- During adenectomy for checking the residual PTH secretion or during surgery of a large goiter.
There are three generations of PTH assays, which measure different circulating forms of PTH . From a global perspective, PTH measurements are now routinely carried out on the the second generation assays.
First generation assays
PTH was measured by radio immunoassay (RIA), using different polyclonal antibodies directed against epitopes within the mid- or carboxy-terminal portion (e.g., 53–84 PTH or 44–68 PTH) of the PTH molecule. In addition to PTH 1–84, RIAs primarily measured PTH fragments. Because of low detection limit and cross-reactivity with PTH-fragments such assays are largely replaced by the second generation assays.
Two-site sandwich assays (IMAs) recognize the intact as well as large C-teminal fragments. They use two different antibodies that are directed against distinct epitopes within PTH (1–84). The capture antibody is directed against an epitope within the C-terminal structure (e.g., epitope region 39–84). The detection antibody (second antibody) is directed against the aminoterminal portion of PTH. Most detection antibodies have their main epitope in region 13–34 of the PTH (1–34) structure, early epitopes like 12–18 and 13–24 being more frequent than distal epitopes 26–32. The detection antibody also binds fragments with a truncated N-terminus such as PTH 7–84. Second generation assays do not detect mid-regional C-PTH fragments such as 53–84 and 44–68, which are detected by first generation assays. The characterization of the second generation assays show that the detection antibody detects not only intact PTH (epitope region PTH 1–34, PTH 2–34) but also C-PTH fragments, non-(1–84) PTH fragments and PTH 7–84.
Third generation assays (bio active PTH 1–84 assay)
These immunometric two-site assays use a similar capture antibody as the second generation assays. The detection antibody is directed against epitopes at the extreme aminoterminal end (region 1–4) of the PTH molecule. These assays do not cross-react with PTH 7–84 and other amino-terminally truncated PTH fragments. In samples from healthy individuals, they measure approximately half of the values as second generation assays, but they only measure the same values as second generation assays if only PTH (1–84) is in the sample .
If a patient’s sample is measured with the second and third-generation assay, the ratio of the results from the bio active PTH assay/intact PTH assay allows the proportion of amino-terminally truncated PTH fragments such as PTH 7–84 to be calculated. These fragments play an important role for CKD patients.
The KDIGO Guidelines recognize a potential benefit of the third-generation assays for patients with CKD, but they recommend that the second-generation assay continue to be used until more exhaustive results are available.
Serum, plasma, in the morning in fasting state: 1 mL
For dialysis patients, the blood sample should be drawn before the dialysis.
Laboratories measure PTH using second-generation assays and specify the results as intact PTH, knowing that not only PTH (1–84) is measured. In the following the abbreviation PTH is used for intact PTH that stimulates the PTH/PTHrP receptor.
PTH is secreted by the parathyroid glands primarily in response to changes in blood ionized calcium (iCa). In cases of hypocalcemia, the normalization of iCa is achieved by the stimulated secretion of PTH via the PTH/PTHrP receptor of the parathyroids. In cases of normocalcemia, the regulation of iCa is mainly regulated by C-PTH fragments. With hypercalcemia, PTH secretion is suppressed and serum PTH levels are decreased. The ratio C-PTH fragments/PTH is increased, because PTH fragments such as PTH (7–84) are elevated.
C-PTH fragments with or without partially contained N-terminus do not stimulate the PTH/PTHrP receptor. If more PTH is required due to increasing hypocalcemia, as is the case with secondary hyperparathyroidism due to chronic kidney disease, vitamin D deficiency or the partial removal of parathyroids, more PTH is secreted and the ratio C-PTH fragments/PTH decreases. The reverse of this is the case with tumor hypercalcemia and vitamin D treatment.
With the use of second-generation PTH assays, besides PTH (1–84) fragmented or truncated PTH structures [e.g., PTH (7–84)] are also measured to a different extent and indicate the finding of an increased PTH concentration. PTH fragments are formed in different amounts in primary and secondary hyperparathyroidism /, /. This particularly plays a role in CKD, when the PTH level is used as a surrogate marker of bone mineral disease (BMD). Due to a cross reaction of PTH with fragmented PTH structures in the second-generation PTH assays, a false impression concerning the BMD status can be mediated .
Hypercalcemia is one of the most common indications for determining PTH. The PTH level allows the differentiation of primary hyperparathyroidism (pHPT) from other causes of the hypercalcemia such as tumor hypercalcemia, vitamin D intoxication, sarcoidosis, and familial hypocalciuric hypercalcemia. Whereas PTH is increased or near the upper reference interval value in 95% of pHPT patients it is within the reference interval for the remaining hypercalcemia.
Usually, the determination of PTH is not required for normocalcemic patients. In older patients with bone complaints and normal calcium levels, mild increase in PTH can be an early sign of pHPT or of secondary hyperparathyroidism (sHPT) due to vitamin D deficiency or CKD.
188.8.131.52 PTH and hypocalcemia
Hypocalcemia is associated with hyperparathyroidism and pseudohypoparathyroidism. In both cases PTH is increased and calcium is decreased.
The blood should be drawn in the morning before 10 a.m. because of mild pulsatility and a circadian rhythm, with higher PTH levels in the evening.
The tests of various manufacturers do not yield comparable values for PTH. Thus, depending on the manufacturer of the kit, the difference for PTH can be up to 3.4 times in the individual sample . According to the KDIGO guidelines an adynamic bone disease exists in CKD patients with PTH levels two times the upper reference interval value. The treatment of these patients with active vitamin D analogues is prohibited. Therefore it is important to always measure PTH using the assay of the same manufacturer.
Intact PTH is a relatively unstable hormone possesing a plasma half life of 2 to 4 minutes therefore the sampling regimen has to pass through a stringent pre-analytical process control. Venous blood sample in EDTAK2 tube is collected and should be subjected to immediate centrifugation at 3,000 g for 15 minutes, frozen and sent in dry ice to the laboratory .
PTH, a peptide of 84 amino acids, is excreted by the parathyroid glands in the form of PTH (1–84), but truncated PTH fragments such as PTH (7–84) are also released. PTH (1–84) is removed from circulation by the liver after being present for 2–4 minutes, cleaved into fragments and excreted through glomerular filtration. For binding the PTH to its receptors, only the first 34 amino acids are required. Only the first 6 amino acids of the N terminus are biologically active.
The ionized calcium (iCa) concentration in the blood is registered by the iCa-sensitive receptor of the parathyroid cells. The high sensitivity of these cells allows them to centrally regulate the calcium content in the circulation. If a hypocalcemia is registered, the parathyroids respond with an enhanced secretion of PTH, which causes the transfer of iCa into the extracellular space due to the following PTH-mediated actions:
- Release of iCa and phosphate from the bone
- Induction of the renal 1,25(OH)2D synthesis, which induces the increased intestinal calcium absorption
- Increase of the renal distal-tubular iCa reabsorption.
The resulting increase of calcium in peripheral blood inhibits the excretion of PTH via feedback inhibition.
The effect of PTH on the tissues is mediated via the PTH/PTHrP receptor a protein of 585–593 amino acids. The receptor binds PTH (1–34) and PTHrP. After binding the receptor stimulates specific G proteins and mediates its signal via two different paths, the cyclic AMP and iCa to the cell nucleus. Refer to .
In PTH stimulated calcium resorption of the bone, PTH does not have a direct effect on mature osteoclasts, but binds to osteoblastic PTH/PTHrP receptors. The osteoblastic cells are activated and secrete cytokines, which stimulate in a paracrine way the differentiation of preosteoclastic cells into active, multi nuclear osteoclasts. PTH also promotes the cell proliferation of osteoblasts and their type-I collagen production and thus also has an osteoanabolic effect in addition to the bone resorption effect.
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The N-terminal sequence of parathyroid hormone-related peptide (PTHrP) has close homology with that of parathyroid hormone (PTH). Some of the physiological functions of PTHrP are similar to PTH because both hormones bind to the PTH/PTHrP-1 receptor. PTHrP acts as an autocrine, paracrine, and endocrine hormone and is able to simulate most of the actions of PTH, including regulation of calcium ion homeostasis, bone resorption, distal tubular calcium reabsorption, and inhibition of proximal tubular phosphate transport .
- Differential diagnosis of hypercalcemia in the case of known or suspected tumor (relative indication)
- Prognostic factor for the development of bone metastases, e.g. breast cancer, small cell lung cancer, renal carcinoma
- Monitoring the course of tumor hypercalcemia
- During bisphosphonate treatment (relative indication).
Radio immunoassay or ELISA using antibodies directed against the aminoterminal (amino acids 1–34) or mid-regional (amino acids 44–68 or 53–84) structures.
Immunometric assays (IRMA)
The two-site IRMA has anti-PTHrP (1–34) as the capture antibody and anti-PTHrP (37–67) as signal antibody. The assay detects PTHrP (1–84). PTHrP (18–34), PTHrP (9–34), PTHrP (1–34), and PTHrP (37–67) do not cross react in the assay .
Principle: PTHrP is enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP is digested with trypsin, and PTHrP-specific tryptic peptide is analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode .
Heparin or EDTA plasma: 1 ml
Hypercalcemia is as a concentration above 10.2 mg/dL (2.55 mmol/L). In 80–90% of the cases, primary hyperparathyroidism (pHPT) or malignant tumors are the underlying cause. Tumor hypercalcemia is characterized by hypercalcemia and hypophosphatemia, two laboratory diagnostic findings which also indicate pHPT. Whereas for pHPT the calcium level is often borderline and is mostly below 11 mg/dL (2.55 mmol/L), the increase with tumor hypercalcemia is significant and is often above 13 mg/dL (3.25 mmol/L) .
Tumor hypercalcemia is characterized by increased bone resorption with release of calcium and is related to the following disorders:
- Osteolytic tumor metastases through the local release of cytokines with cancers of the breast, bladder, lung, uterus, and skin
- The secretion of 1,25 (OH)2D by the tumor
- The formation of PTHrP by the tumor.
- Calcium and PTH are increased with pHPT
- In cases of tumor hypercalcemia, calcium is increased and PTH is low or low normal.
Therefore, from a diagnostic standpoint, the determination of PTHrP is only required if the hypercalcemia cannot be sufficiently clarified based on clinical findings, and calcium, phosphorus and PTH levels in serum/plasma ().
Method of determination
The PTHrP values of different assays are not comparable because they measure different structures. Tests which determine mid-regional structures have values that are 10 times higher than the values measured using aminoterminal assays. Larger structures which encompass the N-terminal and mid-regional structures are recorded using the immunometric assays. PTHrP 1–74, 1–84 or 1–86 are used for calibrating the tests .
Carboxy-terminal structures and, in a smaller percentage, mid-regional structures of PTHrP are excreted renally. In stage 4 and 5 of renal disease, these increase in the plasma.
PTHrP is a protein of 139 amino acids (). PTHrP and PTH are products of two different genes, but are capable of carrying out the same actions on the PTH/PTHrP receptor 1. This is due to similar primary and secondary structures, which are necessary for binding to the receptor. Eight of the first 13 amino acids of the aminoterminal structure are identical. Although the primary structure of PTHrP and PTH between amino acids 18 and 34 is not identical, they have a comparable secondary structure, which allows binding to the receptor. In contrast to PTH, which is only formed by the parathyroids, PTHrP is synthesized in many tissues such as the epithelial, mesenchymal connective tissue, endocrine glands, and the central nervous system. Using the calcium content as a reference, PTHrP does not stimulate the enteral absorption of vitamin D to the same extent as PTH and its effect on the bone is also less. Its physiological endocrine effect is limited to an increase of renal-tubular calcium reabsorption and of the placental calcium transport .
In addition to the above-mentioned endocrine effect, PTHrP also has paracrine/autocrine and intracrine effects . The local autocrine/paracrine effect of PTHrP is that it mediates signals via the PTH/PTHrP receptor like an epithelial factor. In health PTHrP regulates bone development by maintaining the endochondral growth plate. It also plays a role during tooth eruption, development of mammary and regulates the vascular tone via the smooth musculature. The smooth muscles of the uterus and the β-cells of the pancreas also secrete and respond to PTHrP. The intracrine effects relate to the PTHrP influencing the cell cycle via effects on the cell nucleus.
4. Burtis WJ, Brady TG, Orloff JJ, Ersbak JB, Warrell RJ, Olson BR, et al. Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. N Engl J Med 1990; 322: 1106–12.
5. Pandian MR, Morgan CH, Carlton E, Segre GV. Modified immunoradiometric assay of parathyroid hormone-related protein: clinical application in the differential diagnosis of hypercalcemia. Clin Chem 1992; 38: 282–8.
6. Dumon JC, Jensen T, Lueddecke B, Spring J, Barle J, Body JJ. Technical and clinical validation of an immunometric assay for circulating parathyroid hormone-related protein. Clin Chem 2000; 46: 416–8.
8. Hiraki A, Ueoka H, Bessho A, et al. Parathyroid hormone-related protein measured at the time of first visit is an indicator of bone metastases and survival in lung carcinoma patients with hypercalcemia. Cancer 2002; 95: 1706–13.
9. Wada S, Kitamura H, Matsuura Y, Katayama Y, Ohkawa H, Kugai N, et al. Parathyroid hormone related protein as a cause of hypercalcemia in a B-cell type malignant lymphoma. Internal Med 1992; 31: 968–72.
Vitamin D plays an essential role in bone health and in the regulation of calcium and phosphorus metabolism. However, the effects of vitamin D are not limited to mineral homeostasis and maintenance of bone health. Vitamin D physiology extends far above these functions as documented by the presence of vitamin D receptors in many organs and tissues. In addition the 25(OH)D-1α-hydroxylase, the enzyme responsible for the conversion of 25(OH)D (calcidiol) to its biologically active form 1,25 (OH)2D (calcitriol) has been found in other tissues aside from kidneys. The synthesis of calcitriol may be important in regulating cell growth and differentiation via paracrine or autocrine regulatory mechanisms. The action of calcitriol involves a nuclear vitamin D receptor that regulates the transcription of genes in vitamin D target cells that are involved in cell differentiation and calcium homeostasis /, /. The nomenclature of vitamin D precursors and metabolites is shown in .
25-hydroxy vitamin D [25(OH)D]
Anamnestic and clinical advices of vitamin D deficiency are:
- Chronic kidney failure stage ≥ 2 in children and stage ≥ 3 in adults
- Lack of sunlight (seniors)
- Reduced intestinal intake of vitamin D due to fat malabsorption
- Increased metabolism of vitamin D (barbiturates or anti epileptic drugs)
- Increased loss of vitamin D binding protein (nephrotic syndrome, dialysis)
- Hypocalcemia, hypophosphatemia, hyperparathyroidism, increased alkaline phosphatase
- Radiological signs (pseudo fractures, Looser’s reconstruction zones, reduced bone mineral content).
Suspicion of vitamin D overdose or intoxication; increased 25(OH)D concentration.
1,25-hydroxy vitamin D [1,25 (OH)2D]
The plasma concentration of the active vitamin D reflects the calcium content and the renal function. Indications are:
- Chronic renal disease in stages ≥ 3 (not generally recommended by the KDIGO).
Differentiation of hypercalcemias:
- Sarcoidosis, tuberculosis, other granulomatous disease
- Monitoring after medication of 1α-hydroxy vitamin D3 or of 1,25 dihydroxy vitamin D3
- Suspicion of intoxication with hypercalcemia-generating plants, e.g. solanum malacoxylon
- Hypercalciuria of unclear genesis.
Differentiation of hypocalcemia:
- 25(OH)D-1α-hydroxylase deficiency (vitamin D-dependent rickets, VDDR), low calcitriol concentration
- Vitamin D receptor defect (resistance to calcitriol, VDRR, high calcitriol concentration).
Principle: at first the specimen is prepared by precipitation of serum proteins and dissociation of vitamin D from the binding protein. After centrifugation the supernatant is transferred to a solid phase extraction plate and the retained analytes transferred to the auto sampler for chromatographic separation on an ultra-performance liquid chromatography (UPLC) system. The eluent of the UPLC system is introduced in a mass spectrometer set in positive electro spray mode for quantification of 25(OH)D.
Principle: sequential competitive immunoassay format. The specimen is added to a reaction cuvette followed by displacement buffer for removal of vitamin D from the binding protein. Monoclonal antibody conjugated with acridinium ester is added and allows to bind 25(OH)D in the specimen. A 25(OH)D analog conjugated to bovine serum albumin and fluorescein is added along with anti-fluorescein coated paramagnetic particles. The reaction cuvette is washed, and acid and base reagents are added to initiate the chemiluminescent reaction. An inverse relation exists between the amount of 25(OH)D in the specimen and the amount of relative light units detected by the system.
Principle: competitive immunoassay for determining 25(OH)D3 based on the streptavidin-biotin technology. The test uses a polyclonal antibody from sheep against 25(OH)D3, which is ruthenium labeled. The vitamin D3 in the specimen competes for binding with biotinylated 25(OH)D3 antigen which is bound to the streptavidin-coated micro particles.
Serum or plasma: 1 mL
Fasting blood sample in the morning; hyperlipoproteinemia interferes the extraction of vitamin D. For dialysis patients, the blood sample should be taken before the dialysis.
Depending on the geography, 20–50% of the European and North American population have a vitamin D deficiency. The 25(OH)D value reflects the intake of vitamin D with the diet, and its formation in the skin. The determination of 25(OH)D is therefore the best biomarker for assessing the vitamin D status. The synthesis of the active hormone 1,25(OH)2D depends on the stored amount of 25(OH)D and multiple factors, which convert 25(OH)D into 1,25(OH)2D. One important factor is the 25-OHD-1α-hydroxylase (CYP27B1), which converts 25(OH)D into 1,25(OH)2D and its inactivation through the 24-(OH)D hydroxylase (CYP24A1) to 1,24,25 (OH)D.
- Ecological factors (season, local weather conditions, nutrition, sunbathing)
- Unchangeable individual factors (ethnicity, skin pigmentation, age) . These factors especially affect older people in northern latitudes. As they become older, they stay indoors more, wear less exposing clothing, change their eating habits and take in less fat in their diet. Due to increasing atrophy of the skin, fewer pre-vitamin D is converted to vitamin D. Also, due to increasingly limited renal function, the hydroxylation of 25(OH)D to 1,25(OH)2D declines .
The 25(OH)D level depends on the season. The highest values are measured 4–6 weeks after the highest amount of sunlight. The lowest [< 20 μg/L (50 nmol/L)] are measured at the end of winter; from January to April in Central Europe. According to a study , 57% of men and 58% of women in Germany have 25(OH)D values below 20 μg/L (50 nmol/L). For persons 65–79 years of age, the prevalence is 75%.
There is an increased demand for vitamin D in patients with chronic kidney disease, during pregnancy and lactation. A sufficient vitamin D supply for newborns is only achieved if 25(OH)D in the milk of breast-feeding mother is > 30 μg/L (75 nmol/L).
25(OH)D values below 20 μg/L (50 nmol/L) promote secondary hyperparathyroidism . Therefore this concentration has generally been defined as the low threshold value for inhabitants of Central Europe. In part, however, even higher 25(OH)D values, may cause an increase of PTH within the reference interval. People with an optimal vitamin D supply have PTH values below 45 ng/L (4.5 pmol/L). If the vitamin D supply is suboptimal, the PTH level elevates to 45–65 ng/L (4.5–6.5 pmol/L) .
For persons over 70 years of age, secondary hyperparathyroidism can only be reliably prevented with 25(OH)D values above 40 μg/L (100 nmol/L) . Post-menopausal women with 25(OH)D values ≤ 25 μg/L (62,5 nmol/L) often have increased markers of bone resorption . 25(OH)D levels below 5–4 μg/L (12–10 nmol/L) are often associated with osteomalacia. For osteoporosis prevention, 25(OH)D values above 25 μg/L (62.5 pmol/L) are the goal .
Vitamin D supplements are widely recommended for bone health in the general population. In a study the authors tested whether supplemental vitamin D3 (2,000 IU per day)in men ≥ 50 years of age and women ≥ 55 years of age would result in a lower risk of fractures than placebo in the United States. Supplemental vitamin D3, as compared with placebo, did not have a significant effect on total fractures.
Vitamin D receptors are present in most tissues . A deficiency of 25(OH)D can therefore not only be associated with calcium homeostasis disorders and diseases of the bone, but also with the pathogenesis of chronic diseases. Thus, among other things, vitamin D affects the cardiovascular system, the immune system and the glucose tolerance, which means that a 25(OH)D deficiency can be involved with the increased risk of a negative outcome of these diseases. Diseases and conditions with decreased levels of 25(OH)D are listed in . Findings in rickets and osteomalacia are shown in .
In cases of 25(OH)D deficiency and acute or chronic disorders of the bone further investigations are calcium, phosphate, creatinine, 1,25 (OH)2D and PTH in the serum and calcium excretion in the urine. Findings on the differentiation of 25(OH)D deficiency of rickets/osteomalacia are shown in .
The treatment of 25(OH)D deficiency, especially for older patients, serves the purpose of reducing the risk of bone fractures and preventing falling down by improving the neurological/psychological function. The following laboratory examinations are recommended :
- Determination of serum calcium before start of treatment with 1–2 thousand IU of vitamin D, because hypercalcemia is a contraindication
- Measurement of 25(OH)D to control if the dose makes sense. The first measurement should take place 4–6 weeks after treatment is started.
Conditions with increased concentration of 25(OH)D can occur with Vitamin D treatment or corresponding vitamin D3-containing pharmaceuticals (e.g., cod liver oil) or a 25(OH)D treatment.
25(OH)D concentrations above 80 μg/L (200 nmol/L) are only measured in cases with:
- High vitamin D dosage (> 10,000 IE daily)
- Overdose of vitamin D in patients with hypoparathyroidism
- Intoxications with dihydrotachysterol; the intake of these preparations is not analyzed by the assays for 25(OH)D.
1,25(OH)2D (calcitriol) is the active vitamin D and fulfills the functions of a classic hormone . The hormonal signal is transmitted by specific cell receptors of the small intestine, the bone, the kidney, and numerous other organs. In addition to genomically mediated effects, there are also non-genomic effects that act quickly, within 2–6 minutes. The task of 1,25 (OH)2D is to maintain the calcium and phosphate homeostasis in association with PTH and FGF23. In addition, 1,25(OH)2D has pleiotropic effects.
Since 95% of the 25(OH)D in serum circulates as 25(OH)D3, only the 1,25(OH)2D3 is first determined.
The concentration of 1,25(OH)2D3 is 500 to 1,000 times less than that of 25(OH)D, but the affinity to the vitamin D receptor is higher for 1,25(OH)2D.
With the determination of 1,25 (OH)2D disorders in the vitamin D metabolism are diagnosed because the concentration mirrors the activity of the 25-OHD-1α-hydroxylase in the kidney. The compliance of the vitamin D intake can also be monitored. During growth spurts and during pregnancy, high concentrations of 1,25(OH)2D are measured. Hypophosphatemia, hypocalcemia and PTH stimulate the 25-OHD-1α-hydroxylase. This explains an increased formation of 1,25(OH)2D and hyperabsorption of calcium from the intestine for conditions with hypophosphatemia (e.g., for some of the cases of primary hyperparathyroidism or the treatment of rickets with vitamin D).
- The 25(OH)D concentration corresponds with the vitamin D intake and activity
- The half live of 1,25(OH)2D is 4 to 6 hours as compared with a half-life of 2 to 3 weeks for 25(OH)D.
- 1,25(OH)2D concentration may be useful for the diagnosis of genetic rickets, in chronic kidney failure and unexplained hypercalemia, or hypercalciuria.
In fasting state in the morning or before dialysis.
Method of determination
The selection of the assay for the determination of vitamin D, either 25(OH)D or [25(OH)D3 and 25(OH)D2] is important. Since some assays only measure 25(OH)D3, the laboratory must decide whether it will determine 25(OH)D, 25(OH)D3 or 25(OH)D3 and 25(OH)D2 separately. Immunoassays only show a moderate agreement with the LC-MS/MS. Concentrations below 8 μg/L (20 nmol/L) are not reliably measured .
The immunoassays for determining 1,25(OH)2D only use antibodies against 1,25(OH)2D3.
After a heparin injection (e.g., under dialysis treatment) there is an increase of 25(OH)D. The blood sample should be drawn before the dialysis.
At room temperature for 72 hours, decrease of 25(OH)D by 2.3% if the sample is kept in the dark. With exposure to daylight and at room temperature, a decrease by 3.4% after 24 hours and 8.5% after 7 days .
During exposure to solar ultraviolet radiation, 7-hydroxycholesterol is photosynthesized in the skin to cholecalciferol which is converted to vitamin D3. The initial substances of vitamin D3, cholecalciferol and of vitamin D2, ergocalciferol, are lipophilic and are therefore difficult to determine in serum. Their half-life in plasma is 24 hours. Cholecalciferol is formed endogenically and ergocalciferol is taken in with vegetables and fish. Vitamin D3 and vitamin D2 from dietary sources are incorporated into chylomicrons and transported by the lymphatic system into the venous circulation.
Vitamin D in the circulation is bound to vitamin D-binding protein and transported to the liver, where it is converted by vitamin D-25 hydroxylase to 25-hydroxy vitamin D [25(OH)D]. This form of vitamin D is biologically inactive and more than 99% is bound to proteins, especially to vitamin D-binding protein. 25(OH)D is the major circulating form in of vitamin D that is used by the clinicans to determine the vitamin D status.
Approximately 99.7% of total circulating 25(OH)D in serum is bound to vitamin D binding protein and albumin, and only 0.03% is free. It is hypothyzed that only free 25(OH)D can enter cells and induce its biological function.The main proportion is 25(OH)D3 (calcidiol), whereas 25(OH)D2 (ercalcidiol) is primarily measured in a greater concentration in patients with vitamin D2 substitution. The concentration of 25(OH)D3 fluctuates seasonally because the formation of cholecalciferol depends on the effect of sunlight on the skin. The two 25(OH)D molecules differ slightly in terms of their structure, but they have the same biological effect. Therefore it can be important sometimes to measure 25(OH)D3 and 25(OH)D2 to determine the vitamin D status.
Vitamin D (D2 and D3) from the diet and the skin undergo two sequential hydroxylations: first in the liver 25(OH)D2 and 25(OH)D3 and then in the kidneys, leading to its biologically active forms 1,25(OH)2D2 and 1,25(OH)2D3.The half life time of both hormones is 4–6 hours .
25(OH)D is biologically inactive and must be converted in the kidneys by 25-OHD-1α-hydroxylase in the active form 1,25(OH)2D. The main regulators of 25-OHD-1α-hydroxylase activity are the serum concentrations of calcium, phosphate and PTH. The hormones 1,25(OH)2D, PTH and FGF23 regulate by several negative feedback loops the metabolism of phosphate and calcium. The function of the loops results in the enteral absorption and renal handling of calcium and phosphate. In addition the bone mineralization is modulated . Refer to ().
- Enhancement of the intestinal absorption of both calcium and phosphorus. 1,25(OH)2D interacts with the vitamin D receptor (VDR). It is located in the cytoplasm of the cells and belongs to the nucleic receptors of the steroid hormones . After binding of a ligand, the VDR heterodimerizes with the retinoid X receptor (RXR) to form a complex (RXR-VDR), which, after reaching the cell nucleus, binds to vitamin D responsive elements (VDRE). The latter are localized in the promoter region of different genes, whose transcription rates are changed by the binding of the complex RXR-VDR (see example in ). In addition the expression of the epithelial calcium channel and of calbindin 9K, a calcium binding protein, is increased.
- Inhibition PTH synthesis by negative feedback mechanisms and by increased serum calcium levels.
- Regulation of bone metabolism through binding to its receptor on osteoblasts causing an increase in the expression of the RANKL (see ). RANK, the corresponding receptor on preosteoclasts binds RANKL, which induces preosteoclasts to become mature osteoclasts.
- Enhancement of the expression of 25-OHD-24-hydroxylase to catabolize 1,25(OH)2D to the biologically inactive calcitoic acid.
To evaluate the vitamin D status, the concentration of 25(OH)D in serum is measured because it represents the content of vitamin D that is available to the body. If the concentration is normal, the determination of 1,25(OH)2D is not required to asses a relevant vitamin D deficiency if chronic kidney disease in stage ≥ 3 can be ruled out .
Low concentrations of 25(OH)D induce secondary hyperparathyroidism (sHPT) and stimulate the 25-OHD-1α-hydroxylase, which keeps the concentration of 1,25 (OH)2D normal for a long time despite vitamin D deficiency . Thus, a 25(OH)D deficiency primarily causes osteoporosis (decrease in bone mass) as a result of the sHPT. A decrease of 1,25(OH)2D only manifests after a longer period following severe 25(OH)D deficiency. As a consequence of this, a metabolic bone disease results in the form of osteomalacia (i.e.; the reduced synthesis of bone matrix and mineralization defects). In a low vitamin D state, the small intestine absorbs 10–15% of dietary calcium, however when adequate 30–40%.
The maximum bone mass in adolescents depends on the calcium intake, bodily activity, and genetic factors. Up to 85% of the inter individual variability of the bone mass depends on genetic factors, especially on genes that encode the VDR. The VDR gene polymorphism is associated with the mineral mass of the bone. In particular, the polymorphism of the start codon (regulated by splitting the enzyme Fok1) is associated with the bone density. Thus, the Fok1 polymorphism of the VDR receptor appears to predict the bone mineral mass in children .
Vitamin D exists in three types. The majority of 25 (OH)D (88%) and 1,25 (OH)2D (85%) are bound to vitamin D binding protein (DBP). The remaining proportion is bound to albumin with lower affinity. Below 1% of vitamin D exist in the unbound form. The protein bound vitamin D is inactive while the free vitamins exert biological activity. DBP is encoded by three alleles (DBP 1F, DBP 1S, and DBP 2). The DBP phenotype determines the plasma concentration of DPB as well as of 25 (OH)D and 1,25 (OH)2D with the highest concentrations in patients with DBP 1-1, intermediate levels in DBP 2-1, and lowest levels with DBP 2-2 phenotype, respectively .
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6. Roth HJ, Schmidt-Gayk H, Weber H, Niederau C. Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography – tandem mass spectrometry as a reference. Ann Clin Biochem 2008; 45: 153–9.
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Biochemical bone markers are a tool in the diagnosis and monitoring of metabolic bone disease . Their most application in clinical practice is monitoring of osteoporosis and the efficacy of anti resorptive treatment as an adjunct to bone mineral density measurements. Other applications are the use of these markers for bone diseases other than osteoporosis, such as bone mineral disorders (chronic renal failure, vitamin D deficiency, hyperparathyroidism), hypercortisolism, hyperthyroidism, diabetes mellitus type I, metastatic bone disease, multiple myeloma, and Paget’s disease.
Bone formation markers
- The bone isoform of alkaline phosphatase (bone ALP), see
- Osteocalcin, see
- N-terminal pro peptide of type 1 collagen (P1NP), see
Bone resorption markers
These markers are direct or indirect products of the osteoclasts. The cross links of the collagen molecules of the bone matrix form special structures during the synthesis or degradation that are used for analysis ().
Because most of the metabolic bone diseases are characterized by an increase in bone resorption, these markers are of special interest. Important markers are:
- The pyridinium cross-links pyridinoline (PYD) and deoxypyrodinoline (DPD); see .
- The N- and C-telopeptides of type 1 collagen; see .
- The tartrate-resistant acidic phosphatase type 5b (TRAP-5b).
Bone is a composite material of about 70% mineral matter (mainly hydroxyl apatite), 5–8% water and 22–25% organic matrix (mainly type I collagen) . Collagen type I is a heterodimer made up of three polypeptide chains (triple helix) and is rich in the amino acids lysine and proline. Two polypeptide chains (α1) are identical, the third (α2) has a different amino acid sequence. After synthesis, two α1chains and one α2 chain are twisted to one triple helix, the fibril. The three α chains form a triple helix by covalent binding via hydroxylated lysine remnants of the adjacent chains ().
The α chains are synthesized as pre procollagen and consist of a signal peptide at the N-terminal end as well as N- and C-terminal pro peptides. After the pro peptides are cleaved from the fibril, it is transported to the extracellular space via the Golgi apparatus.
The fibrils arrange as bundles in the extracellular space to produce large collagen fibers. The carboxy-terminal and aminoterminal ends of pro collagen I are enzymatically cleaved during extracellular processing and fibril formation prior to incorporation of type I collagen into the bone matrix.
When type I collagen is degraded by osteoclasts during the resorption process, NTX and CTX fragments of type 1 collagen are released in the circulation. During the degration of collagen, NTXs and CTXs are relatively specific for bone tissue breakdown. Other tissues comprised of type 1 collagen are not actively metabolized by osteoclasts, and therefore different fragments are formed during breakdown of non skeletal tissue. Immunoassays, which use antibodies against CTX are called β-crossLap tests (CTX).
Post translational processing of lysine and hydroxylysine residues to form Pyr and D-Pyr cross links are essential for stabilizing the mature forms of collagen fibers and elastin. Both cross links are present in bone and absent from most tissues including skin. D-Pyr is more bone specific because Pyr cross links are present in cartilage and several soft tissues. Three-fold more Pyr is present in bone compared to D-Pyr.
In metabolic bone diseases, the remodeling process is accelerated and, consequently, the concentration of bone turnover markers in serum and urine is increased. The bone turnover markers reflect the total rate of bone formation and bone resorption of the skeleton. Bone turnover marker allow a dynamic assessment of bone metabolism, thus supplementing the static information of the bone mass, which is determined by measuring the bone mineral density .
The intraindividual variation of the bone turnover markers is relatively high, which means that bone turnover markers are only recommended to a limited extent for diagnosing bone diseases, in particular if it involves the determination in urine. The currently essential clinical application is the monitoring of metabolic bone disease, especially osteoporosis under anti resorptive treatment . The biomarker should be determined before, after a 3–6 month period, after 12 months, and then annually. Bone resorption markers significantly decrease as early as 3–6 months. As a rule, a decline of 30–70% in comparison to the initial value is expected, then it will reach a plateau. An insufficient decrease is a sign of failed treatment or poor compliance of the patient. Using bone mineral density measurement changes in density are normally observed after 1–2 years .
The bone turnover markers provide no information about the current bone mineral content or bone fragility, nor do they provide information about a specific site with increased bone turnover in the skeleton. This is only possible using the bone mineral density measurement, because it allows the bone mass to be assessed as normal, osteopenic, or osteoporotic. Because bone mineral density provides information of the current bone status and bone markers provide information about current bone metabolism, both parameters complement each other, and provide comprehensive information about patients bone status and bone metabolic activity . Thus, patients with a high rate of bone resorption have an increased fracture risk and those with a combination of low bone mineral density and a high rate of bone resorption have the highest risk of fracture .
The reference intervals of the bone turnover markers have limited authority in comparison to other biomarkers in clinical chemistry. Thus, for post-menopausal osteoporosis, the bone marker levels of most patients are within the reference intervals and only relative changes compared to a value before treatment play a role. To overcome this problem and to allow the detection of physiological changes in bone marker values in an individual the use of least significant change is used ().
Least significant change (LSC): the LSC is the minimal change of a bone turnover marker which must be occur during treatment to make a response significant. The LSC is calculated from the intraindividual and analytical variability according to a formula . A change in bone markers that is higher than the LSC would have a 95% chance of being too high in comparison to normal biological variability. Thus the LSC for β-CTX measurements is 31.2% for a change in bone mineral density of the spinal column of 3.8% .
Evaluation of bone turnover markers
The bone turnover markers are not recommended for diagnosing osteoporosis and for determining the bone mass. The currently best established and most common clinical application of bone turnover markers is the monitoring of osteoporosis treatment and controlling the compliance of these patients .
Bone formation markers
Bone formation markers, especially the bone ALP, have advantages over resorption markers in assessing the bone participation in cases of vitamin D deficiency, hyperparathyroidism, osteomalacia/rickets, renal osteodystrophia, osteoblastic metastases, and Paget’s disease. P1NP is intended for assessing the anabolic treatment of osteoporosis with recombinant parathyroid hormone.
Bone resorption markers
In the treatment of osteoporosis, the resorption markers, particularly pyridinolines and C-telopeptides (CTX), have become significant. Experience shows, however, that in most cases (e.g., post-menopausal women, older men) the CTX determination (EDTA plasma at 8 a.m. in fasting state) is the superior marker for bone resorption and pyridinolines move into the background. In contrast, for tumor patients (e.g., bronchial carcinoma, breast carcinoma, multiple myeloma) the pyridinolines are the superior marker, determined in the first morning urine.
6. Anlinker M, Bieglmayer C, Dimai HP, Gasser RW, Kudlacek S, Obermayer-Pietsch B, et al. Labordiagnostik in der Prävention, Differentialdiagnose und Verlaufskontrolle der Osteoporose. J Lab Med 2009; 33: 140–6.
The bone specific ALP (BALP) is produced by the osteoblast and is deposited as buds in cell membrane vesicles. BALP plays an important role in the bone formation. Approximately 20–40% of serum ALP is BALP (see also ).
Indicator of global bone formation activity:
- Monitoring of bone mineral disease in patients with chronic renal failure
- Management of patients with Paget’s disease
- Monitoring of bisphosphonate therapy for osteoporosis
- Management of patients with bone metastases.
Serum or heparin plasma, no EDTA, citrate or oxalate plasma: 1 mL
To evaluate discrete changes of the bone metabolism, such as post-menopausal, in cases of chronic kidney disease-mineral bone disease, or after administration of glucocorticoids (e.g., after an organ transplantation) the BALP is often determined in addition to the ALP.
In one study the ALP increased by 24% after menopause and the BALP by 77%. Expressed as Z-score (multiple of the standard deviation above the mean value for pre-menopausal women), the ALP increased by 0.9 and the BALP by 2.2. The BALP responds relatively slow as bone formation marker.
For monitoring CKD-MBD the KDIGO guidelines recommend the ALP or BALP be determined in stages G4 - G5D every 12 months or more frequently if PTH is increased. In comparison with a healthy control group, patients with stage G4 and G5D had the values shown in Ref. and for BALP and other bone markers. Regarding the response of BALP to treatment of renal osteodystrophia see .
An increase in BALP or ALP is only seen after more than 6 months of vitamin D deficiency. When the vitamin D deficiency is corrected, the BALP increases even further in the first weeks, but then returns to normal over the course of 6–12 months.
In the first 5 days, there is a decrease of BALP in the serum, followed by an increase, which is still not normalized for large fractures after 100 days and often only reach the initial values after one year .
Carcinomas metastasizing in the bone, especially carcinomas of the breasts, lungs and prostate can cause an increase of BALP . The prevalence is shown in . The metastases lead to dysregulation of the bone metabolism and cause osteolytic, osteoblastic or mixed bone metastases, depending on the malignoma. In cases with osteoblastic metastases, there is an excessive bone formation with an increase of BALP. In patients with prostate carcinoma and bone metastases, resorption markers quickly respond to treatment. The BALP, if at all, only shows signs of decline after more than 4 weeks . With multiple myeloma, the function of osteoclasts is particularly increased, therefore, it does not make sense to determine bone formation markers such as BALP .
Administering sodium fluoride: if sodium fluoride is administered to treat osteoporosis, an increase of the BALP occurs (NaF stimulates the osteoblasts).
11. Kress BC, Mizrahi IA, Armour KW, Marcus R, Emkey RD, Santora AC II. Use of bone alkaline phosphatase to monitor alendronate therapy in individual postmenopausal osteoporotic women. Clin Chem 1999; 45: 1009–17.
12. Overgaard K, Alexandersen P, Riis BJ, Christiansen C. Evaluation of a new commercial IRMA for bone-specific alkaline phosphatase during treatment with hormone replacement therapy and calcitonin. Clin Chem 1996; 42: 973–4.
Hilmar Stracke, Lothar Thomas
OC (bone gamma-carboxylglutamic acid-containing protein; GLA) is synthesized by the osteoblasts during the matrix mineralization phase and is incorporated into the organic bone matrix . A small fraction of newly synthesized OC is secreted in the circulation and, in individuals with normal renal function, excreted in the urine. During bone resorption OC is degraded, however up to 70% also enter the circulation. Because OC in circulation may be both newly synthesized during bone formation and released during resorption, there is some question whether OC should be considered a marker of osteoblast activity or an indicator of bone matrix metabolism or turnover . Its synthesis is regulated by 1,25(OH)2D.
Monitoring bone remodeling in:
- Osteoporosis (assessment of the bone turnover)
- Carcinoma with bone metastases
- Primary hyperparathyroidism
- Renal osteopathy.
Immunometric assay, enzyme-linked immunosorbent assay (ELISA). Most commercially available assays use antibodies which detect N-terminal and mid-regional fragments of the OC (N-MID osteocalcin) . There is no OC reference preparation. The patient values obtained using various test kits are not comparable .
Serum, EDTA or lithium heparin plasma; blood sampling at 8–9 a.m. in fasting state: 1 mL
* Depending on the test kit
In clinical diagnostics OC is used a bone formation marker and the serum level reflects 10–40% of the OC that is not incorporated in the bone matrix, but is released into the circulation as intact 1–49 OC. Released OC is rapidly metabolized, one third is retained as an intact molecule, one third is a large N-terminal fragment of the OC (amino acids 1–43), and one third exists as a mid-regional fragment and in the form of smaller fragments .
An increased serum concentration of OC is the result of an enhanced bone remodeling. Quantitative histomorphometric examinations and examinations of the kinetics and balance of calcium have shown that OC is more a biomarker of bone formation than of bone resorption .
The OC fragments are eliminated by metalloproteinases of the liver and kidney. Since OC fragments are excreted renally, an increased OC concentration can also be caused by limited renal clearance.
Increases of OC in serum are mostly related to an enhanced bone turnover, as with primary hyperparathyroidism, Paget’s disease, and high-turnover osteoporosis.
The OC concentration in the serum is, like all bone metabolism markers, higher in children than in adults. In children, OC correlates to the growth rate, therefore there is a peak during puberty. Whereas, for example, CTX are relatively consistently higher in post menopausal than in premenopausal women, there is a temporary increase for OC in the time following menopause. In premenopausal women, there are significantly higher OC levels during the luteal phase .
One advantage of OC in comparison to the bone-specific ALP is in the stronger and faster reaction when glucocorticoids are administered (suppression of osteoblast activity). Disadvantages are dependency on the glomerular filtration rate, weaker response with Paget’s disease, and limited bone formation specificity. The clinical significance of OC in comparison to ALP is shown in
Since the OC gene expression is regulated directly by 1,25(OH)2D and corticosteroids, the OC values of patients who are treated with these medications should be interpreted with caution.
Blood collection is recommended in the morning in fasting state. There is a circadian rhythm of the OC with high values in the early morning and a nadir in the afternoon and early evening. Then an increase follows, which reaches its maximum value from midnight to 4 a.m. .
Anticoagulated blood samples have excessively low OC values in comparison to the serum. In one study the level was only 83.6% of the level in serum when Li-heparin was used, 65.1% for EDTA, 70.4% for sodium citrate (0.13 mol/L) and 37.3% for oxalate/fluoride.
Method of determination
In addition to intact OC the N-terminal and mid-regional fragments (N-MID-OC) are also measured.
OC formation is stimulated by physiological levels of 1,25(OH)2D. Parathyroid hormone and the glucocorticoids prednisolone and deflazacort suppress the 1,25(OH)2D stimulated OC synthesis. OC is important for the synthesis of clotting factors of the prothrombin complex and other vitamin K-dependent proteins. Dicumarol suppresses the OC synthesis.
Since higher bone resorption rates are observed in winter with lower supplies of vitamin D and a vitamin D deficiency also stimulates the osteoblasts, the highest OC values are observed in February and the lowest in July.
N-MID-OC: in whole blood, at room temperature (RT) for 8 hours, at RT for 24 hours in serum, for 48 hours at RT 1 week at 4–8 °C, and for 1 year at –20 °C in EDTA plasma.
OC is a protein composed of 49 amino acids and has a MW of 5.8 kDa; it is formed from a larger 11 kDa precursor by proteolytic cleavage and contains up to three γ-carboxyglutamic acid residues (bone gla protein).
The precursor consists of three structures:
- A signal peptide made up of 23 amino acids, which is cleaved after translation
- The target peptide of 26 amino acids, which directs the peptide to the γ-carboxylation
- The OC protein of 49 amino acids. The OC is one of the three vitamin K-dependent proteins, which are formed from the osteoblasts. The others are the matrix Gla protein and protein C. Vitamin K is an essential cofactor of the post-translational γ-carboxylation of OC. Through carboxylation, a second carboxyl group is introduced to the glutamyl residues in the positions 17, 21 and 24 during the formation of γ-carboxy glutamyl residues (Gla) (). This modification leads to a conformational change of the protein with an increase of the affinity for calcium and hydroxyl apatite .
OC is the largest portion of the non collagenous proteins of the organic bone matrix. It is considered to be a specific marker of osteoblast function and of osteoid mineralization, since it is exclusively synthesized by osteoblasts. The synthesis is stimulated by vitamin D.
The overwhelming portion of the OC is incorporated into the bone matrix after its release from the osteoblasts. A small proportion enters the circulation. Circulating OC has a half-life of 4 min. and is primarily eliminated via the kidney. In the presence of a GFR below 30 [mL × min–1 × (1.73 m2)–1], the plasma OC level exceeds the reference interval. Similar interactions are observed, when the OC concentration is set in relation to the renal plasma flow.
The OC concentration rises with age. Women have higher serum levels than men. OC is not only present in bone and serum but is also found in extraosseous calcifications during the early mineralization phase and in atherosclerotic plaques.
4. Eastell R, Delmas PD, Hodgson SF, Eriksen EF, Mann KG, Riggs BL. Bone formation in older normal women: concurrent assessment with bone histomorphometry, calcium kinetics, and biochemical markers. J Clin Endocrinol Metab 1988; 67: 741–8.
14. Naylor KE, Eastell R. Measurement of biochemical markers of bone formation. In: Seibel MJ, Robins SP, Bilezikian JP, eds. Dynamics of Bone and cartilage metabolism. San Diego; Academic Press 1999; 401–10.
Collagen type I (also known as collagen alpha, COL1A1, and alpha-1 type I collagen) is the largest component of fibrillar collagen found in cartilage and connective tissues. It is synthesized by fibroblasts, osteoblasts, and odontoblasts and has a theoretical molecular weight of 138 kDa.The bone matrix is comprised up to 90% of collagen type 1, which is synthesized by osteoblasts. The cleavage of collagen type 1 yields two relative large extension peptides termed pro collagen 1 carboxy-terminal pro peptide (PICP) and pro collagen 1 aminoterminal pro peptide (PINP). In practice, serum PINP has been found to reflect histomorphometric measures of bone formation and has been identified as the most promising marker of bone formation and designated the reference marker of bone formation in osteoporosis by the International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine .
The procollagen type 1 N-propeptide is a trimeric peptide of the molecular weight of about 35 kDa, consisting of two type 1 prokollagen-alpha-1 chains and a prokollagen-alpha-2 chain which are bonded non-covalently. Prokollagen type 1 molecules are synthesized by osteoblasts, and the pro-peptide extensions at the amino- and carboxy-terminals (PINP and PICP, respectively) are cleaved off and laid down to form the osteoid matrix during bone formation. Since the PINP and PICP molecules are produced in equimolar amounts with collagen-1 molecule, their concentrations in the circulation might be expected to reflex bone formation rate .
- Evaluation of progress and therapeutic monitoring of the osteoporosis
- Vitamin D deficiency-caused secondary hyperparathyroidism
- Assessment of the mineral bone disease in chronic kidney disease (CKD-MBD).
Principle: the sample is incubated with a biotinylated antibody and then a second ruthenium labeled antibody is added to the preparation, together with streptavidin coated micro particles. A sandwich complex is formed, which binds to the micro particles through biotin-streptavidin. The micro particles are magnetically bound to the surface of an electrode. Applying a voltage to the electrode induces a chemiluminescent emission, which is measured using a photomultiplier and is transformed into a concentration value via a calibration curve.
EDTA plasma, heparin plasma, serum: 1 ml
A distinction is made between intact P1NP (trimeric form) and the monomer form. The natively formed trimeric form with a half-life of 10 hours is converted into the monomer form in vivo.
Post menopausal women without osteoporosis have serum P1NP levels of 19–100 μg/L (central 95% range), the mean value and the standard deviation are 47.7 ± 19,9 μg/L . For women with post menopausal osteoporosis, the concentrations are in a comparable magnitude (50.5 ± 18,6 μg/L).
In a further study 74% of the post menopausal women over 35 years of age with osteoporosis had somewhat higher mean P1NP values than premenopausal women without osteoporosis and 32.5% had concentrations above the upper reference interval value. Both studies show that the diagnosis of the post menopausal osteoporosis with P1NP is only possible in a few cases.
- The concentrations of P1NP increased for 83% of the patients treated with rPTH and
- 88% of the patients treated with bisphosphonate (alendronate) showed a decrease in comparison to the initial value
- A least significant change (LSC, see ) of 20% served as a change criterion.
A further study on treating post menopausal women with rPTH (teriparatide) has shown that an increase of P1NP in the first 3 months after the start of treatment has the highest diagnostic sensitivity for predicting an improvement of bone mineral density (BMD) of the spine in the following 18 months. The improvement was defined as an increase of the bone mineral density above 3% .
EDTA plasma or heparin plasma should be preferred.
Method of determination
Tests which only measure the trimeric form yield lower values than those that also detect monomer P1NP.
At room temperature 24 hours, at up to 8 °C 5 days, 6 months at –20 °C.
2. Brandt J, Krogh TN, Jensen CH, Fredriksen J, Teisner B. Thermal instability of the trimeric structure of the N-terminal propeptide of human procollagen type 1 in relation to assay technology. Clin Chem 1999; 45: 47–53.
5. Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, et al. The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 2003; 349: 1207–15.
6. Chen P, Satterwhite JH, Licata AA, Lewiecki EM, Sipos AA, Misurski DM, et al. Early changes in biochemical markers of bone formation predict BMD response to teripartide in postmenopausal women with osteoporosis. J Bone Mineral Res 2005; 20: 962–70.
Post translational processing of lysine and hydroxylysine residues to form the pyridinium cross links pyridinoline (PYD; hydroxylysylpyridinoline) and deoxypyridinoline (DPD; lysylpyridinoline) are essential for stabilizing the mature forms of collagen and elastin. Both cross links are absent from most tissues and are found in bone. PYD and DPD are liberated from bone matrix by osteoclasts and about 60% is bound to protein and the remaining 40% is free . Because most metabolic bone diseases are characterized by an increase in bone resorption, these biomarkers are of special interest.
- Post menopausal osteoporosis
- Primary hyperparathyroidism
- Assessment of mineral bone disease in cases of chronic kidney disease (CKD-MBD)
- Paget’s disease
- Tumor-associated hypercalcemia
- Carcinoma with bone metastases
Evidence of a treatment effect:
- 6 months after the start of hormone substitution
- 3 months after the start of bisphosphonate treatment
- 1 month after the start of parenteral bisphosphonate and/or parathyroid hormone treatment
A distinction is made between the assays of total (t) and free (f) pyridinolines in the urine. The former are determined with the HPLC, the latter with HPLC or immunoassays.
PYD and DPD by means of HPLC
- Acid hydrolysis in order to release the protein-bound cross links
- Partition chromatography using cellulose
- HPLC separation using reversed phase principle. The measurement is undertaken fluorometrically with PYD and DPD being separately determined in a single run (). Omitting acid hydrolysis allows the free fraction of cross links to be determined.
This method measures the free fraction of DPD. The determination is preformed by competitive enzyme immunoassay using solid-phase immobilized monoclonal antibodies and enzyme-conjugated DPD. By acid hydrolysis of the sample before starting the immunoassay the sum of the free and peptide-bound fraction of PYD and DPD can also be determined by immunoassay .
This method measures the free and protein-bound fraction of PYD. DPD shows 100% cross reactivity. The determination is performed using a competitive enzyme immunoassay with solid-phase immobilized PYD and enzyme-conjugated polyclonal antibodies.
Spot morning urine without additives: 5 mL
Since PYD and DPD are only present in mature collagen type 1 and not in the collagen of the skin, the renal secretion of PYD and particularly of DPD can serve as a specific biomarker for the quantitative assessment of bone resorption. In healthy individuals, 15–85% of total PYD (tPYD) and total DPD (tDPD) are present in the urine in free form as fPYD and fDPD. The remainder is bound to peptides from the bone collagen.
The premenopausal range is seen as a reference interval of the pyridinolines. Post menopausal DPD values above 60 μg/g creatinine are interpreted as increased bone resorption. Approximately 30–40% of the post menopausal women have values above this threshold.
Since the osteoclasts mainly release crosslinked telopeptides and other peptides from the bone collagen, tPYD and tDPD increase post menopausally more rapidly than fPYD or fDPD.
tPYD and tDPD also decrease more rapidly than fPYD and fDPD during hormone substitution after menopause . The more enhanced the bone resorption, the less the proportion of fDPD, and as bone resorption increases, the proportion of fDPD becomes less. It is true that tDPD and fDPD correlate , but the evidence of fDPD becomes less important in comparison to the total excretion due to the decreasing proportion of the fDPD with increasing total excretion. The same applies to the measurement of tPYD and fPYD. Therefore the measurement of tPYD and tDPD is favored .
In the first morning urine, post menopausal women have higher excretion than during the day, which is even more accentuated in post menopausal women with osteoporosis () . In women with osteoporosis, bone resorption increases especially at night. Apparently, the bodily activity during the day leads to bone resorption approaching normal.
In addition to the daily rhythm, there is a monthly and annual rhythm of tDPD, at least in premenopausal women () . This figure also shows that, for a low 25(OH)D in February, mild regulatory hyperparathyroidism and mild increased bone resorption occurs.
For patients with lung carcinoma and radiologically proven bone metastases, pyridinolines (tPYD and tDPD) are sensitive markers . However, patients without bone metastases and patients with benign lung diseases also show some signs of increased excretion. Thus, the diagnostic sensitivity is high, but the specificity is low. In patients with lung carcinoma or breast carcinoma without bone metastases, increased values of tPYD and tDPD are often found because, presumably, the tumors produces substances such as PTHrP that cause increased bone resorption.
In tumor patients, the bone metastases are shown with more sensitivity by tPYD and tDPD than by NTX and CTX. This may be due to the fact that, in tumor patients, the degradation of collagen no longer occurs regularly as in post menopausal women through cathepsin K, but irregularly through, for example, matrix metalloproteinases, so that structures other than the telopeptide of collage type 1 occur.
Due to the seasonal rhythm with the vitamin D deficiency phase during the winter in Central and Northern Europe, the pyridinolines, like all markers, lose their diagnostic effect. Due to vitamin D substitution for tumor patients during the winter, the pyridinolines and other bone resorption markers can gain in importance for the detection of bone metastases /, /.
The disadvantage of tPYD and tDPD in comparison to serum markers is the relation to creatinine excretion if spot urine is investigated.
A reason as to why the tDPD excretion relative to creatinine can be increased in post menopausal women is that they are less physically active than younger premenopausal women. This causes muscle mass and urine creatinine to decrease more sharply than the bone mass, which results in an increased ratio of tDPD/creatinine. tDPD/creatinine is disproportionately increased with hyperthyroidisms as well . One cause is the increase of muscle metabolism.
Hormone replacement therapy (HRT)
During HRT, the serum level of the sexual hormone-binding globulin (SHBG) increases ; this is a result of the passage of estradiol through the liver. SHBG binds estradiol and testosterone equally, therefore the concentration of free testosterone in women decreases under oral HRT. The reduced free testosterone leads to a decline in muscle mass, the creatinine excretion is therefore lower and the ratio of tDPD/creatinine turns out higher. Since the β-CTX (see ) are measured in the EDTA plasma, they indicate the bone resorption in women under HRT more correctly than the ratio of tDPD/creatinine. Thus, in all of the post menopausal women with HRT there was a decrease of the β-CTX in relation to the values of premenopausal women. This was not the case, however, for all HRT women when the ratio tDPD/creatinine was measured.
In another series of women under various forms of HRT, it was seen that low-dose transdermal patches (25 μg estradiol administered per day) led to normal tDPD/creatinine ratio in the first morning urine. It is documented that this form of HRT does not lead to an increase in SHBG. Therefore, the muscle mass will not decrease and the creatinine excretion in the urine also does not decrease.
These patients with well functioning kidneys excrete little creatinine in the urine, which is why the ratio of tDPD/creatinine turns out too high.
With Paget’s disease and osteoporosis, bisphosphonate treatment leads to a sharp decrease of tPYD and tDPD, while fPYD and fDPD do not decrease . Vitamin D treatment in older women with vitamin D deficiency leads to a sharp decrease of CTX and a less pronounced decrease of tPYD and tDPD, while fPYD and fDPD do not decrease . The fDPD is thus not suitable for either the monitoring of the bisphosphonate treatment or the vitamin D treatment.
Method of determination
Most of the experiences are present in the first morning urine. It provides the following advantages:
- The urine is more concentrated than in later samples
- The peaks of the pyridinolines are significantly higher due to the higher degradation rates at night
- The sample must not be exposed to direct sunlight, because pyridinolines are fluorescing structures that can be destroyed by long-term exposure to UV light.
- Intraindividual variation: tPYD 71 (57–78)%, tDPD 67 (53–75)%
- Day-to-day variation: tPYD 16 (12–21)%, tDPD 16 (5–24)%
- Inter individual variation of premenopausal women: tPYD 26 (12–63)%, tDPD 34 (8–98)%
- Inter individual variation of post-menopausal women: tPYD 36 (22–61)%, tDPD 40 (27–54)%.
5. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinium crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 1991; 6: 639–44.
6. Garnero P, Gineyts E, Arbault P, Christiansen C, Delmas PD. Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion. J Bone Miner Res 1995; 10: 641–9.
8. Aoshima H, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, and Inoue T. Circadian variation of urinary type I collagen cross-linked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks. Bone 1998; 22: 73–8.
9. Woitge HW, Knothe A, Witte K, Schmidt-Gayk H, Ziegler R, Lemmer B, Seibel MJ. Circannual rhythms and interactions of vitamin D metabolites, parathyroid hormone, and biochemical markers of skeletal homeostasis: a prospective study. J Bone Miner Res 2000; 15: 2443–50.
10. Muley Th, Herb KP, Tuengerthal S, Schmidt-Gayk H, Ebert W. Markers of bone metabolism do not prove bone metastases in lung cancer patients. CECHTUMA 2003; 4th Central European Conference on human tumor markers. Karlovy Vary, Czech Republic, Abstract, 2003.
13. Kamel S, Brazier M, Rogez JC, Vincent O, Maamer M, Desmet G, Sebert JL. Different responses of free and peptide-bound cross-links to vitamin D and calcium supplementation in elderly women with vitamin D insufficiency. J Clin Endocrinol Metab 1996; 81: 3717–21.
14. Vesper HW, Demers LM, Eastell R, Garnero P, Kleereoper M, Robins SP, et al. Assessment and recommendations on factors contributing to preanalytical variability of urinary pyridinoline and deoxypyridinoline. Clin Chem 2002; 48: 220–35.
15. Rauch F, Schönau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994; 102: 94–7.
During bone resorption, N-telopeptide (NTX) and C-telopeptide (CTX) of type 1 collagen are released into circulation (). NTX and CTX are relative specific biomarkers for the degradation of bone. None-bone tissues comprised of type 1 collagen form different fragments during degradation . Therefore NTX and CTX have an optimal bone specificity under these preconditions. In addition both telopeptides are important bone resorption markers and are the largest fraction indicating increased bone resorption and lead to significant elevations in serum and urine in comparison to PYD and DPD.
The CTX are preferred in comparison to DPD in clinical laboratory diagnostics because:
- CTX occur as α-CTX (no isomerization of asparagine acid) and β-CTX (isomerized asparagine acid). Isomerization is a spontaneous process which occurs slowly in the post-translational period. Therefore, the β-form is only present in mature tissues such as bones and β-CTX tests only detect collagen fragments from mature type 1 collagen. Newly formed collagen is not determined, which is why the β-CTX test is not a bone formation marker .
- The β-CTX test is also called β-CrossLaps assay. β-CTX can be determined using immunoassays on automated platforms with good precision in the serum and urine.
Determination of the bone resorption:
- Estimation of an increased regular bone turnover (e.g., post menopausal)
- Monitoring the treatment for osteoporosis
- Monitoring the bone turnover of dialysis patients
- Monitoring the treatment of malign bone disease.
Two-site immunoassay specifically for the crosslinked β-isomerized type-1 collagen telopeptide fragments. Two monoclonal antibodies are used, each of which records the antigen Glu-Lys-Ala-His-βAsp-Gly-Gly-Arg (CrossLap-antigen) .
Principle: the specimen is incubated in a tube with a biotinylated antibody and then a second ruthenium-labeled antibody is added, together with streptavidin-coated micro particles. A sandwich complex is formed, which binds to the micro particles through biotin-streptavidin. The micro particles are magnetically bound to the surface of an electrode. Applying a voltage to the electrode induces a chemiluminescent emission, which is measured using a photomultiplier. The emission is transformed into a concentration via a calibration curve.
EDTA blood, urine: 2 mL
In clinical practice, measurements of either serum or urine β-CTX are used in the diagnosis and management of a spectrum of metabolic and malignant bone diseases. β-CTX is released in the circulation in remodeling of the bone in high amounts and in remodeling of the skin to a small proportion. Therefore β-CTX has developed as a sensitive and specific biomarker of bone turnover (. Because of its high sensitivity for the degradation of type 1 collagen, the possibility for the determination in serum and the adaption to automated analyzers has favored β-CTX as a widely used bone resorption marker .
Day to day variability
Serum β-CTX levels show low variability.
A single meal within 60–120 min. before blood collection can decrease the serum β-CTX concentration by up to 50%.
Hormone replacement therapy
Preparing the patient for blood sampling
- 12 hours of abstinence from food, before blood collection. After waking up, only water is allowed until the blood sample is taken
- Blood collection between 7 a.m. and 9 a.m. in fasting state
- If it is not possible to draw blood before 9 a.m., the recommended alternative to β-CTX determination is to determine pyridinolines in morning spot urine
- For dialysis patients, the blood sample should be taken before the dialysis.
EDTA plasma is better suited than serum, because the stability of β-CTX is greater.
Stability of β-CTX
In the EDTA whole blood 6 hours at room temperature (RT). In EDTA plasma 24 hours at RT, 48 hours at 4 °C, and 1 month at –20 °C. In serum at RT 12–24 hours.
6. Reginster JY, Henrotin Y, Christiansen C, et al. Bone resorption in post-menopausal women with normal and low BMD assessed with biochemical markers specific for telopeptide derived degradation products of collagen type 1. Calcif Tissue Int 2001; 69: 130–7.
7. Iki M, Morita A, Ikeda Y, et al. Biochemical markers of bone turnover predict bone loss in perimenopausal women – the Japanese Population-based Osteoporosis (JPOS) Cohort Study. Osteoporos Int 2006; 17: 1086–95.
9. Garnero P, Sornay-Rendu E, Claustrat P, Delmas PD. Biochemical markers of bone turnover, endogenous hormones and the risk of fractures in postmenopausal women: the OFELY study. J Bone Miner Res 2000; 15: 1526–36.
11. Rosen CJ, Chesnut III CH, Mallinak NJ. The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab 1997; 82: 1904–10.
12. Guillaume J, Lafage-Proust MH, Souberbielle JC, Lechevallier S, Deleaval P, Lorriaux C, et al. Severe secondary hyperparathyroidism in patients on haemodialysis is associated with a high initial serum parathyroid hormone and beta CrossLaps level: Results from an incident cohort. PLOS One 2018; 13 (6): e0199140
15. Qvist P, Christgau S, Pedersen BJ, Schlemmer A, Christiansen C. Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting. Bone 2002; 31: 57–61.
16. Schmidt-Gayk H, Spanuth E, Kötting J, Bartl R, Felsenberg D, Pfeilschifter J, Raue F, Roth HJ. Performance evaluation of automated assays for α-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone (Biorose multicenter study). Clin Chem Lab Med 2004; 42: 90–5.
DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; IL-1, interleukin-1; IL-6, interleukin-6; M-CSF, macrophage-colony stimulating factor; PG, prostaglandin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α
* The repetition of these tests during the winter months (January to April) is recommended. Fasting morning (7:30 - 8:30 AM) blood sampling is best. For urine tests such as pyridinolines approximately 10 mL of first morning urine.
BD, bone disease; reduced, amount of non-mineralized osteoid increased
Definition of CKD-MBD
Definition of renal osteodystrophy
Rickets and osteomalacia
* Deoxypyridinoline or other resorption markers
** In cases of primary calcium deficiency within the framework of initial renal osteodystrophia.
Calcidiol, 25(OH)D; VDDR, vitamin D-dependent rickets, disturbed 1α-hydroxylase; VDRR, vitamin D-resistant rickets, disturbed calcitriol receptor; VDDR and VDRR are also called PVDR = pseudo-vitamin D deficiency rickets type I and II. DPD, deoxypyridinoline; N, normal; NI, not indicated
DPD, deoxypyridinoline; OM, osteomalacia; TIO, tumor-induced osteomalacia; XLH, X-chromosomal hypophosphatemia.
Increased PTH secretion due to parathyroid adenoma or carcinoma
Reactively increased PTH secretion due to renal or intestinal-related calcium deficiency
Autonomic hyperplasia of the parathyroids with increased PTH after many years of secondary HPT
* Excretion in the 24-hour urine; ** resorption marker: pyridinoline (PYb), deoxypyridinoline (DPD), β-CrossLaps (CTX), NTX, tartrate-resistant acid phosphatase (TRAP-5b). Comment: The calcitriol values are only elevated for primary HPT if there is a sufficient concentration of 25(OH)D (calcidiol).
↓ Decreased or reduced; ↑ Elevated or increased; PTH, parathormone; GFR, e.g., determined by creatinine clearance or cystatin C; EH test, Ellesworth-Howard test, PTH injection; Hypo P, hypoparathyroidismd; PHP, pseudohypoparathyroidism: pseudo PHP, pseudo-pseudo hypoparathyroidism; N, normal; Ni, no increase; Sn, subnormal
Data expressed in mg/dL (mmol/L), * Values expressed as x ± s for a collective of only 7–19 children in each case, other references express: for 0–1 month 3.9–6.0 mg/dL (1.0–1.5 mmol/L) and for 1–6 months, 3.7–5.9 mg/dL (0.95–1.5 mmol/L). Conversion: mg/dL × 0.2495 = mmol/L. The reference intervals of ionized Ca and albumine-adjusted calcium are identical for assessing the calcium status.
The Payne formula is based on the observation that each 1 g/dL reduction in albumin < 4.0 g/dl results in an approximately 0.8 mg/dL (0.2 mmol/l) reduction in total calcium. Payne RB, Carver ME, Morgan DB. Interpretation of total calcium: effects of adjustment for albumin concentration on frequency of abnormal values and on detection of change in the individual. J Clin Pathol 1979; 32: 56–60.
Clinical and laboratory findings
Clinical and laboratory findings
Conversion: mg × 0.02495 = mmol
Clinical and laboratory findings
Clinical and laboratory findings
Data expressed in mg/dL (mmol/L).
Conversion: mg/dL × 0.3229 = mmol/L
Clinical and laboratory findings
Clinical and laboratory findings
Clinical and laboratory findings
CKD stages 4–5D: ALP every 12 months, or more frequently in the presence of elevated PTH
AHO, Albright’s hereditary osteodystrophy ; PHP Ia, pseudohypoparathyroidism type Ia; PPHP, pseudo-pseudohypoparathyroidism; PHP Ib, pseudohypoparathyroidism type Ib; Gsα, subunit of the guanyl nucleotide-binding protein; ↓, decreased; ↑, elevated; +, present; –, absent; N, normal
Clinical and laboratory findings
Conversion 25 (OH)D: ng/ml × 2.5 = nmol/L
Conversion 1,25 (OH)2D: ng/L × 2.6 = pmol/L
Clinical and laboratory findings
1 Gender-related hypophosphatemia; 2 Autosomal dominant hypophosphatemic bone disease; 3 Autosomal recessive vitamin D-dependent rickets type I; 4 Vitamin D-dependent rickets type II (hereditary resistance to calcitriol). PTH, parathyroid hormone; P, phosphate; * 25-OHD-1α-hydroxylase.
n, normal; ↑ increased; ↑↑ greatly increased; ↓ decreased; ↓↓ greatly decreased.
Values expressed as mean value ± SD or median and 95% CI, BMD, bone mineral density
HPT, hyperparathyroidism; N, within the reference interval; ↑, significantly increased; ↓, significantly decreased
Values are 5th and 95th percentiles in Ref. and x ± s in Ref. . Determination with HPLC after acid hydrolysis. The conversion factor of PYD μg/g creatinine in μmol/mol creatinine is 0.263. The conversion factor of DPD μg/g creatinine in μmol/mol creatinine is 0.278.
Clinical and laboratory findings
Figure 6.1-1 Sequence of bone remodeling, modified according to Ref. . 1. Resting bone with lining cells that contain osteocytes. 2. The lining cells recede and the underlying membrane is removed by metalloproteinase. 3. Osteoclasts are recruited and activated followed by fusion to become multi nucleated osteoclasts. 4. Osteoclasts digest the underlying bone forming a resorption cavity. 5. Osteoblasts are recruited to the cavity. 6. Osteoblasts lay down new osteoid, which is then calcified.
Figure 6.1-2 Osteoblast differentiation starting from the stem cell (SC), modified from Ref. . The stem cell differentiates into the osteoblast (OB) via the mesenchymal stem cell (MSC) and the pre-osteoblast (PreOB). Paracrine, endocrine and autocrine factors with a stimulating effect: endothelial growth factor, EGF; insulin growth factors, IGFs; bone morphogenetic protein-2, BMP-2; transgrowth factor-β, TGF-β; vascular endothelial growth factor, VEGF; fibroblast growth factor-2, FGF-2; parathyroid hormone, PTH.
Figure 6.1-3 Mechanisms of osteoclast activation by cytokines, macrophage colony-stimulating factors (M-CSF) and Receptor Activator of Nuclear factor-kappa B Ligand (RANKL) formed by osteoblasts and stromal cells. Osteoprotegerin (OPG) is an inhibitor of osteoclast formation.
Figure 6.1-4 Collagen synthesis from two α1-chains and one α2-chain. Markers of the collagen formation are carboxy-terminal pro collagen type I pro peptide (PICP) and N (amino)-terminal pro collagen type I pro peptide (PINP). Collagen degradation and the markers of collagen resorption (CTX, C-terminal cross links; NTX, N-terminal cross links) are shown. With kind permision from Ref. .
ALP, alkaline phosphatase; BALP, bone ALP isozyme; BSP, bone sialo protein; CTX, C-terminal crosslinks; ICTP, collagen type 1 C-terminal telopeptide; NTX, N-terminal crosslinks; PICP, carboxy-terminal procollagen type 1 propeptide; PINP, N-terminal procollagen type 1 propeptide; TRAP, tartrate-resistant acidic phosphatase.
Figure 6.2-1 Relationship between calcium excretion in the 24-hour urine and dietary intake of calcium . The daily calcium intake is displayed on the abscissa and the calcium excretion is displayed on the ordinate. 10 mmol corresponds to 400 mg.
Figure 6.2-4 Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor (PTHSR) or of the calcium-sensitive receptor (CaSR), modified according to Ref. . The Gs-protein acts in the cellular signal transmission as an on-off switch.
1) The Gs-protein consists of the subunits α, β and γ. In the resting state, all three proteins are non-covalently bound to each other and the α-unit has bound GDP. The switch position is “Off”.
2) If the membrane-bound PTHSR or CaSR is activated by the binding of PTH or calcium (Ca), it interacts with the Gs-protein. This results in the dissociation of GDP from the receptor and GTP is bound.
3) The binding of GTP leads to the dissociation of the Gs protein in the α- and βγ-subunits. The α-unit binds to the effector, which possesses adenylate cyclase activity and cleaves ATP into cyclical AMP and Pi. The switch position is “On”.
4) The α-unit (a guanosin triphosphate), which is bound to the effector, hydrolyzes GTP to GDP. This inactivates the α-subunit and allows it to combine again with the βγ-subunit. Hence, the switch is in “Off” position.
Overall, by this process, PTH or Ca, the “first messengers”, release cyclic AMP, the “second messenger” and thus transmit a signal into the cell.