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Examining Calcium, Magnesium & Phosphorus Levels in Tissues via Ultrafiltration Probes, Lecture notes of Physiology

University of Central Oklahoma (UCO)Physiology

A study that utilized ultrafiltration probes to measure calcium, magnesium and phosphorus concentrations in bone, muscle and subcutaneous tissue in freely moving sheep. The investigation compared calcium, magnesium and phosphorus concentrations in different tissues and in blood, and analyzed the data using a specific model. The study verified the in vitro recoveries of calcium, magnesium and phosphorus and reported the results.

What you will learn

  • What were the calcium, magnesium and phosphorus concentrations in different tissues and in blood?
  • How were calcium, magnesium and phosphorus concentrations measured in bone, muscle and subcutaneous tissue?

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In vivo sampling using membrane
probes has provided a tool for inves-
tigating the chemistry of the intersti-
tial space. It has provided data which
have expanded the understanding of
both physiological and pathological
conditions. Under a contract with
NASA, we developed ultrafiltration
probes which could be implanted in
bone, muscle and subcutaneous tis-
sue to sample the interstitial fluid. In
this project, sheep were used as the
animal model. This permitted the in-
vestigation of calcium, magnesium
andphosphorusconcentrationsinlo-
cations not previously accessible to
directinvivomeasurement. The goal
of this project was to develop tools
to study mineral metabolism. These
tools will facilitate the development
of a better understanding of bone
physiology and pathology.
Calcium, magnesium and phos-
phorus are the minerals present in
the largest quantities in bone and are
importantingivingstrengthand
shape to the bone. However, they
have important physiological func-
tions in other parts of the body as
well.Calcium isanessentialelement
in intercellular regulation and meta-
bolism. Extracellular calcium is an
essential component of cofactors re-
quired for bone formation, blood
clotting, adhesions molecules, and
as a first messenger in signaling
functions (1). Many of its actions
within cells are dependent on a con-
stant extracellular pool of available
calcium. The parathyroid hormone-
Vitamin D-calcitonin system and the
parathyroid gland “set point” tightly
regulate extracellular calcium con-
centrations. Magnesium is involved
in neuromuscular transmission and
is a cofactor in various enzyme reac-
tions. It is important in ribosomal
protein synthesis and ATP energy
transfer. Phosphorus is ubiquitous
throughout the body. Phosphates are
a major component of the blood
buffering system. Phosphorus is
found in DNA, RNA, proteins and
phospholipids and is important in
energy metabolism. It therefore af-
fects all aspects of physiology (2).
One of the benefits of membrane
probe sampling is that different tis-
sues can be sampled simultaneously.
This provides a tool to study the dif-
ferences in distribution of these min-
erals. In this study, calcium,
magnesium and phosphorus concen-
trations were compared in the differ-
ent tissues and in blood.
Calcium exists in blood in three
forms: protein bound, complexed
and ionized. Magnesium is also
found in the blood in the protein
bound or ionized forms. Complexed
and ionized calcium can pass though
the probe membrane but protein
bound calcium cannot. The recov-
ered calcium is called the ultrafilter-
able calcium. Ultrafiltrate
membrane probes can also be used
in vitro to separate protein bound
calcium and magnesium from other
forms of these minerals in blood.
Interstitial Fluid Calcium,
Magnesium and Phosphorus
Concentrations in Bone, Muscle
and Subcutaneous Tissue Sampled
with Ultrafiltration Probes
Elsa M. Janle
1
, Ph.D.,
Melony Cregor
1
, BS, Janice
Sojka
2
, VMD.
1. Bioanalytical Systems, Inc.
2701 Kent Avenue
West Lafayette, IN
47906-1382
2. School of Vet. Medicine,
Purdue University
West Lafayette, IN
47907-1248
Specialized ultrafiltration probes make it possible to measure calcium,
magnesium and phosphorus in bone, muscle and subcutaneous tissue in
freely moving sheep.
81 Current Separations 19:3 (2001)
pf3
pf4
pf5

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In vivo sampling using membrane probes has provided a tool for inves- tigating the chemistry of the intersti- tial space. It has provided data which have expanded the understanding of both physiological and pathological conditions. Under a contract with NASA, we developed ultrafiltration probes which could be implanted in bone, muscle and subcutaneous tis- sue to sample the interstitial fluid. In this project, sheep were used as the animal model. This permitted the in- vestigation of calcium, magnesium and phosphorus concentrations in lo- cations not previously accessible to direct in vivo measurement. The goal of this project was to develop tools to study mineral metabolism. These tools will facilitate the development of a better understanding of bone physiology and pathology. Calcium, magnesium and phos- phorus are the minerals present in the largest quantities in bone and are important in giving strength and shape to the bone. However, they

have important physiological func- tions in other parts of the body as well. Calcium is an essential element in intercellular regulation and meta- bolism. Extracellular calcium is an essential component of cofactors re- quired for bone formation, blood clotting, adhesions molecules, and as a first messenger in signaling functions (1). Many of its actions within cells are dependent on a con- stant extracellular pool of available calcium. The parathyroid hormone- Vitamin D-calcitonin system and the parathyroid gland “set point” tightly regulate extracellular calcium con- centrations. Magnesium is involved in neuromuscular transmission and is a cofactor in various enzyme reac- tions. It is important in ribosomal protein synthesis and ATP energy transfer. Phosphorus is ubiquitous throughout the body. Phosphates are a major component of the blood buffering system. Phosphorus is found in DNA, RNA, proteins and phospholipids and is important in

energy metabolism. It therefore af- fects all aspects of physiology (2). One of the benefits of membrane probe sampling is that different tis- sues can be sampled simultaneously. This provides a tool to study the dif- ferences in distribution of these min- erals. In this study, calcium, magnesium and phosphorus concen- trations were compared in the differ- ent tissues and in blood. Calcium exists in blood in three forms: protein bound, complexed and ionized. Magnesium is also found in the blood in the protein bound or ionized forms. Complexed and ionized calcium can pass though the probe membrane but protein bound calcium cannot. The recov- ered calcium is called the ultrafilter- able calcium. Ultrafiltrate membrane probes can also be used in vitro to separate protein bound calcium and magnesium from other forms of these minerals in blood.

Interstitial Fluid Calcium,

Magnesium and Phosphorus

Concentrations in Bone, Muscle

and Subcutaneous Tissue Sampled

with Ultrafiltration Probes

Elsa M. Janle^1 , Ph.D., Melony Cregor^1 , BS, Janice Sojka^2 , VMD.

  1. Bioanalytical Systems, Inc. 2701 Kent Avenue West Lafayette, IN 47906-
  2. School of Vet. Medicine, Purdue University West Lafayette, IN 47907-

Specialized ultrafiltration probes make it possible to measure calcium, magnesium and phosphorus in bone, muscle and subcutaneous tissue in freely moving sheep.

81 Current Separations 19:3 (2001)

Materials and Methods

Probe Development Ultrafiltration probes are small implantable devices consisting of semipermeable hollow fiber mem- branes attached to a conducting tube. The fibers were implanted in the tis- sue to be sampled. The tubing exited through the skin and was attached to a needle hub. The needle was in- serted into a VACUTAINER™, which created a negative pressure within the probe. The semiperme- able fibers do not allow substances with a molecular weight higher than 40,000 daltons through their pores. Small molecules and ions pass though the membrane while large molecules such as proteins are ex- cluded. The probes developed for this project were modifications of the BAS large animal probe (PN MF-7028). All probes in this study were constructed with three loops of semi-permeable fiber with a 40, molecular weight cut-off. Each fiber was 12 cm long. The bone probe ( F1 ) had a reinforcing sheath to prevent kinking of the tubing as it made a 90° bend when exiting the bone. It also had an additional porous cuff at the end of the sheath to promote tissue ingrowth and increase positional sta- bility. Additional suture retainers were added to anchor the probe in place and prevent dislodging with the normal movement of the sheep. For the muscle and subcutaneous probes, only one cuff was used, but additional suture retainers were added for anchoring. In Vitro Recoveries. Before the probes were used for in vivo studies, in vitro recoveries were performed for calcium, magnesium and phos-

phorus to verify that they would cross the membrane (3). In vitro re- coveries were: calcium 100%±3%, magnesium 99%±7% and phospho- rus 102%±4%. Sheep. Five female mixed-breed sheep (age range 1 to 4 years old) were obtained from the Purdue Sheep Farm. Protocols for this study were approved by the Purdue Ani- mal Care and Use Committee. Each sheep was surgically implanted with bone, muscle and subcutaneous ul- trafiltration probes. Free choice ac- cess to mixed grass/alfalfa hay and water was available throughout the experimental period. Probe Implantation. A detailed description of the probe implanta- tion procedure has been published previously (4). The sheep were anes- thetized and maintained under isoflurane general anesthesia during the implantation procedure. Strict aseptic technique was used for all surgical procedures. For bone probe implantation, a hole was drilled into the medullary cavity via the greater trochanter. A second hole was drilled into the medullary cavity at the distal portion of the shaft of the femur. A looped wire was then inserted into the proximal hole and, using a guide in the distal hole, directed out of the femur. A length of suture was at- tached to the wire and the wire was withdrawn. The suture was then af- fixed to the probe tubing and the probe was carefully drawn into place. When the procedure was fin- ished, the UF probe fibers were in the marrow cavity of the femur with the collection tubing exiting from the great trochanter, and then sub- sequently from the skin.

For implantation into the mus- cle, the probe was placed into a curved introducer. Skin incisions were made at the entrance and exit points to facilitate placement. The introducer was inserted through the skin and about 7 cm into the quadri- ceps muscle. The curved introducer penetrated the body of the muscle and curved back to the second inci- sion. The introducer was pulled out, leaving the probe in place. The sub- cutaneous probe was inserted by a similar procedure. However, a straight introducer was used since the placement was immediately un- der the skin. After placement of the probes, the incisions were sutured, and a suture was placed around the probes to hold them in place until the tissue ingrowth into the cuff could anchor them in place. Needle hubs were then placed on the ultrafiltra- tion probes and the needles were in- serted into Vacutainers. Fitted jackets were put on the sheep, and the Vacutainers were placed into pockets in the jackets. Sample Collection. Samples were collected continuously from the UF probes. The Vacutainers were changed at least once a day. During weekdays, they were changed twice a day. Samples were aliquoted im- mediately and frozen at -80° C until assayed. Heparinized blood samples were collected twice a week in the morning when the ultrafiltrate tubes were changed. Samples were centri- fuged and plasma aliquoted and stored at -80° C until assayed. Sample Analysis. Plasma and ul- trafiltrate samples were analyzed for calcium by spectrophotometric analysis using the o-cresolphthalein complexone method from Sigma Di- agnostic Kit No. 587. In plasma this analysis yields the total calcium, which includes protein bound cal- cium, complexed calcium and ion- ized calcium. In the ultrafiltrate samples, this assay measures the sum of ionized and complexed cal- cium. In order to determine the com- plexed and ionized calcium in plasma, the plasma samples were ul- trafiltered using U 3-2 probes. Ion-

F The Bone Ultrafiltration Probe has three 12 cm looped ultrafiltration fibers. A reinforcing sheath prevents kinking of the tubing at the bone exit site. Two tissue ingrowth cuffs and suture retainers help to stabilize the position of the probe.

www.currentseparations.com 82

was significantly greater than mus- cle and subcutaneous interstitial magnesium (p < 0.01). There was no difference between muscle and sub- cutaneous interstitial magnesium. In all tissues the interstitial magnesium was significantly higher than the plasma ultrafilterable magnesium (p < .01) ( F4 ).

Phosphorus Concentrations The mean concentrations of phosphorus in bone, muscle and sub- cutaneous tissue were 4.30 mg/dL, 4.35 mg/dL, and 4.45 mg/dL, re- spectively. There were no significant differences between any of the tis- sues. Interstitial phosphorus in each of the tissues was significantly lower than the plasma phosphorus (6. mg/dL, p < 0.0001).

Discussion

Homeostasis in multi-celled organ- isms is achieved by maintaining the concentration of ions, cell constitu- ents, and water within cells at a con- stant level. This, in turn, is maintained by homeostatic mecha- nisms that ensure the cells are bathed in extracellular fluid that has con- stant concentrations of many con- stituents (5). The extracellular calcium and magnesium concentra- tions are not identical in all tissues, however. There are clear examples of subcompartments within the larger extracellular pools, which dif- fer in concentration from the blood. Extracellular calcium levels are dif- ferent from blood in many tissues including kidney, bone, lung, gastro- intestinal tract and skin. The extreme example would be the fluid in the resorptive lacunae of the bone that

may reach calcium concentrations of 76 mM (6). Direct sampling of the extracellular fluid is necessary to de- termine calcium and magnesium concentrations at different sites, as they are not reflected accurately by sampling the vascular space. Knowl- edge of the extracellular calcium and magnesium concentrations in a given tissue is particularly important in studies where perturbations may not be reflected in the blood concen- trations, such as in disease states or in conditions of microgravity. The changes in extracellular concentra- tions within tissues and compart- ments are unknown in these instances. The ultrafiltrate probe is the ideal tool to investigate interstitial fluid chemistry in both large and small animals (3,4,7-13). It has the advantage that it can be used for long periods without loss of recovery (7,8). Previously, most ultrafiltration sampling had been done from subcu- taneous tissue, although muscle in horses (11) and spine in dogs (10) have also been sampled. In this study, we demonstrated that the tech- nique is also useful in studying bone and muscle in sheep. Sheep were used as a model of bone research because of their low cost, docile dis- positions, and large bones. The sheep in this study were all older. This is an advantage when compar- ing them to humans, as older animals have H aversian remodeling. Ovariectomized ewes show de- creased bone mass along with in- creased biochemical markers of bone turnover. Despite these advan- tages, sheep are not a widely used animal in mineral research, and questions about how they resemble human conditions remain. This study demonstrated that there were differences in the rela- tionship of plasma and interstitial bone mineral concentrations for the different bone minerals. For cal- cium, the concentration of ultrafil- terable calcium is less in the interstitial fluid of bone, muscle and subcutaneous tissue than in the plasma ( F2 ). For magnesium, the

F Figure 4. In all tissues the interstitial magnesium was significantly higher that the plasma ultrafilterable magnesium (p < .01). This pattern is the reverse of what is found for calcium.

T Magnesium concentrations in plasma, and bone, muscle and subcutaneous interstitial space.

Bone Muscle Subcutaneous Plasma Total

Ultrafilterable

2.25 ± 0. n= 1.73 ± 0.04 1.58 ± 0.04 1.62 ± 0.03 1.20 ± 0. n=124 n=177 n=208 n=

F Calcium in plasma exists in three forms: protein bound, complexed and ionized. The total calcium represents the sum of these three forms. Ultrafilterable calcium represents the sum of ionized and complexed calcium.

0.

1.

2.

3.

4.

5.

6.

7.

8.

9.

Calcium (mg/dL)

Plasma Bone Muscle Subcutaneous

Protein Bound Ionized Complexed

0

0.

1

1.

2

2.

Magnesium (mg/dL)

Plasma Bone Muscle Subcutaneous

Total UF

www.currentseparations.com 84

situation is reversed ( F3 ) and the in- terstitial magnesium in all tissues studied was greater than the plasma ultrafilterable magnesium. Variations in concentration from one tissue to another also differed for the various bone minerals. For phos- phorus, there were no significant dif- ferences among the interstitial concentrations of the different tis- sues studied. For magnesium, bone interstitial magnesium was signifi- cantly greater than subcutaneous and muscle interstitial magnesium. For calcium, the relationship was re- versed and bone interstitial calcium was lower than the other tissues, al- though the difference was signifi- cant only for muscle. Although there were significant differences among ultrafilterable calcium concentra- tions in the different tissues, the ion- ized calcium concentrations were not different. The fact that there is a difference in UF calcium in different tissues, but not in ionized, is consis- tent with homeostatic control of ion- ized calcium, which is to be expected since ionized calcium is the physi- ologically active form. The com- plexing agents may be acting as a buffering system to help maintain constant ionized calcium concentra- tions. Capillary ultrafiltration probes were shown to be a useful tool for

monitoring chemical dynamics in the intercellular fluid. These probes have potential applications in the de- velopment of anti-osteoporosis drugs and countermeasures to mi- crogravity-induced bone loss. They will also be useful tools in nutritional studies and in the study of bone physiology and pathology.

Acknowledgements

This work was funded by NASA grant NAS9-97020. The authors wish to thank Zonda Birge, Mollie Ranaletta and Renu Bajaj for their technical assistance.

References

  1. Garong WF. A review of Medical Physiology. 16th ed. Apppleton and Lang, Norwalk CT, 1993, p348.
  2. Berne RM and Levy MN. Physiology. 2nd ed. The CV Mosby Co, St. Louis, p876, 1988.
  3. Janle EM, Sojka J. Use of Ultrafiltration Probes in Sheep to Collect Interstitial Fluid for Measurement of Calcium and Magnesium. Contemporary topics in Laboratory Animal Science, 39:46-50, 2000.
  4. Sojka J, Janle EM, Adams S, Rhode C. Collection of Interstitial Fluid From Bone and Muscle Using Ultrafiltration Probes. Journal of Investigative Surgery, 13: 289-294,
    2. Guyton AC. Textbook of Medical Physiology. 7th ed. WB Saunders, Philadelphia, PA, p382-392, 1986.
    3. Guyton AC. ibid. p941-942.
    4. Janle-Swain E, Van Vleet J, Ash SR. Use of a Capillary Filtrate Collector for Monitoring Glucose Levels in Diabetics. American Society of Artificial Internal Organs Transactions, 33: 336-340, 1987.
    5. Janle EM, Ostroy S, Kissinger P. Monitoring the Progress of Streptozotocin Diabetes in the Mouse with the Ultrafiltration Probe. Current Separations, 11: 17-19,
    6. Janle EM, Kissinger PT, Pesek JF. Short interval monitoring of glucose in Zucker Diabetic Fatty (ZDF) rats. Current Separations, 14: 58-63,
    7. Robertson JT, Huffmon GV, III, Brannon TL, Leffler CW, Gunter BC, White RP, Prostaglandin Production After Experimental Discectomy. Spine 21: 1731-1736,
    9. Spehar AM, Tiedje LJ, Sojka JE, Janle E, Kissinger PT. Recovery of endogenous ions from subcutaneous and intramuscular spaces in horses using ultrafiltrate probes. Current Separations 17: 47-51, 1998.
    10. Janle EM, Kissinger PT. Monitoring physiological variables with membrane probes. Acta Astronautica, 43: 87-99, 1998.
    11. Janle EM, Swain SL, Hucker E, Ash SR. Long Term Monitoring of Glucose with the Capillary Filtrate Collector. Diabetes 40, Suppl 1, 427A, 1991.

85 Current Separations 19:3 (2001)