parathyroid

1. The long-term effects of parathyroidectomy on Bone histology and coronary
artery calcification in hemodialysis patients: prospective observational study
Osama El-Shahat, Ibrahim El-Zayat, Ahmed Halawa, Hamed Eleraky, Nadia Amr El-Husseini,
Waleed Hassan, Maria Yassen, Florence Lima, Marie Claude Monier-Faugere, Hartmut Malluche
Introduction
Chronic kidney disease-Mineral bone disorder (CKD-MBD) including vascular
calcification, biochemical abnormalities; and renal osteodystrophy increases
morbidity and mortality of dialysis patients.1, 2
Secondary hyperparathyroidism
(SHPT) starts in the early stages of CKD and represents one of the major metabolic
abnormalities in dialysis patients. With dialysis initiation, most patients have
hyperplasia of the parathyroid glands3
and noticeably elevated parathyroid
hormone (PTH) levels4
, which tend to reach higher levels with longer dialysis
vintage. 5, 6
Many observational studies reported that high PTH levels are
associated with increased mortality in this population. 7–14
Since SHPT increases
the chance of renal osteodystrophy and cardiovascular calcification15
, it requires
proper monitoring and timely control of PTH.

2. The medical treatment modalities for SHPT includes oral intestinal phosphate
binders, oral or intravenous (IV) calcitriol or active vitamin D analogs, and
calcimimetic agent either oral (cinacalcet) or IV (etelcalcetide).
Parathyroidectomy (PTX) is an effective treatment option of intractable SHPT
whenever the skillful surgery and nephrology teams are well-trained and available.
Medical treatments are much more expensive and even less available especially in
low and middle-income countries in which PTX could be an appropriate option for
management of severe and resistant SHPT.
The main indications for PTX in dialysis patients are severe uncontrolled
hyperparathyroidism, persistent hypercalcemia and/or hyperphosphatemia.17
Successful PTX lower serum PTH, serum phosphorus, serum calcium. Yajima A et
al. reported that PTX suppresses the bone resorption and enhances bone formation
one week after PTX.17
However, PTX may cause hypoparathyroidism and
excessive reduction of bone turnover, which is associated with poor outcomes in
dialysis patients, including vascular calcification.18
Long-term effect of PTX has not been widely studied yet. Bone turnover status is
worthy to be evaluated in a large number of patients for long duration after PTX.

3. Aim of the study: to evaluate the long-term effects of PTX on:
1- Bone turnover and other bone histomorphometric parameters.
2- Coronary artery calcifications and other cardiovascular calcification parameters.
3- Noninvasive CKD-MBD serum biomarkers.
Inclusion criteria:
1- CKD-5D on regular hemodialysis for at least 6 months.
2- Age of 18 years or older.
3- Serum iPTH level above 1200 pg/dl.
Exclusion criteria:
1- Active malignancy.
2- Liver failure or active liver disease.
3- Active infection.
4- Incompetent patients who are not able to give consent.
5- Any medications that can affect or interact with bone histology as
bisphosphonates, calcitonin, steroid, denosumab, estrogen and fluoride in the
last 6 months.
6- Presence of positive deferoxamine test.

4. 7- Serious gastrointestinal disease, ethanol or drug abuse, chronic
inflammatory disease.
8- Pregnancy or breast-feeding.
9- Corrected serum calcium concentration of less than 7.5 mg/dl.
Study design:
This is a twelve months longitudinal prospective study of 50 patients who will be
subjected to PTX for severe uncontrolled secondary or tertiary HPT. Clinical,
laboratory, multi-slice computed tomography (MSCT) and bone biopsy will be
performed at the time of PTX (baseline) and 12-month thereafter. During the
follow-up, doses of calcitriol, phosphate binders and elemental calcium from
calcium salts will be recorded.
Biochemical parameters:
The following blood biochemical parameters will be determined: (1) iPTH and
whole PTH; (2) BSAP (3) PINP, (4) TRAP-5b, (5) RANK-L and OPG, (6)
Sclerostin, (7) FGF-23, (8) Soluble Klotho, (9) Activin-A, (10) Fetuin-A and (11)
CTX.
(1) iPTH and whole PTH Assays: iPTH will be measured by a
radioimmunometric assay (RIA) (Scantibodies, Santee, CA): normal range is 14 to

5. 66 pg/ml; intra- and inter-assay coefficients of variation are <5 and <7%,
respectively.
(2) Bone specific alkaline phosphatase (BSAP): Serum BAP levels will be
measured using an enzyme immunoassay (EIA) (Metra BAP EIA (Quidel San
Diego, CA). Normal serum BAP levels range are 18-75 U/L; intra- and inter-assay
coefficients of variation are <6% and <8%, respectively.
(3) Procollagen type I N-terminal propeptide (PINP): Total P1NP levels will be
measured using an ELISA (USCNK, Wuhan, China); the intra- and interassay
coefficients of variation are ,9% and ,10%, respectively.
(4) Tartrate resistant acid phosphatase 5b (TRAP-5b): levels will be determined
using an EIA (MicroVue,/Quidel, Santa Clara, CA). Normal serum levels range are
1.2-6.7 U/L; intra- and inter-assay coefficients of variation are <2.2% and <3%,
respectively.
(5) Receptor activator of nuclear factor kappa ligand (RANK-L) and
Osteoprotegerin (OPG): Levels will be determined using an enzyme-linked
immunosorbent assay (ELISA) (Alpco, Windham, NH). The normal range for
RANK-L is 1–50 pmol/L and the intra- and inter-assay coefficients of variation are
<5% and <9%, respectively. The normal range for Osteoprotegerin (OPG) is 1–30

6. pmol/L and the intra- and inter-assay coefficients of variation are <10%,
respectively.
(6) Sclerostin: Serum sclerostin levels will be measured using an EIA
(Tecomedical Group, Sissach, Switzerland). The intra- and interassay coefficients
of variation are,3.1% and 3.5%, respectively.
(7) FGF-23: Serum intact FGF-23 levels will be measured using two-site
immunosorbent assay (ELISA) kit (Kainos Laboratories, Tokyo, Japan). Two
specific murine monoclonal antibodies bind to full-length FGF-23. One antibody is
immobilized onto the microtiter plate well for capture. The other antibody is
conjugated to horseradish peroxidase (HRP) for detection. Normal range for
FGF23 is 18-108 pg/mL. The kit has a high sensitivity with the minimum detection
limit of 3 pg/mL as well as a wide quantification range of 3-800 pg/mL. Each
sample will be assessed in triplicate. With concentrations > 800 pg/mL, the
procedure will be repeated using a 1/10 dilution.
(8) Klotho: Serum Klotho will be measured using enzyme-linked ELISA kit
(Immuno Biological Laboratories Co., Ltd., Japan). Mean values for the inter- and
intra-assay coefficients of variability are 6.5% ± 3.3% and 3.6% ± 1.5%,
respectively.

7. (9) Activin-A: Activin-A levels will be measured by ELISA, R&D Systems Inc,
Minneapolis, USA—sensitivity 3.67 pg/mL, intra- and inter-assay coefficient
variation of 4.2-4.4 % and 4.7–7.9 %, respectively.
(10) Fetuin-A: Serum fetuin-A will be measured using a quantitative sandwich
enzyme immunoassay technique (R&D Systems Inc MINN), with reported intra-
and inter-assay coefficient variations of 4.0 and 8.4 %, respectively, and sensitivity
0.62 ng/ml.
Bone Biopsies:
Bone biopsies will be performed in all patients at baseline and 12 months later.
Different tetracycline compounds (tetracycline hydrochloride and demeclocycline
hydrochloride) will be used to distinguish timed bone labels. Tetracycline
hydrochloride gives a yellow color and demeclocycline hydrochloride
(Declomycin®, Lederle, Wayne, NJ), a golden-yellowish fluorescence under
fluorescent light microscopy. The labeling schedule consists of a 2-day oral
administration of tetracycline hydrochloride (250 mg bid) followed by a drug-free
interval of 10 days and subsequent oral administration of demeclocycline
hydrochloride (150 mg bid) for 4 days. The bone biopsy will be performed 2-4
days after the last demeclocycline dose. This dosing regimen is adjusted for CKD-
5D patients. The investigators will provide study participants with a calendar

8. indicating the date of the bone biopsy and dates on which the compounds should
be taken. She will also call study participants on the respective dates to remind
them to take the medication. Study participants will undergo local anesthesia using
Lidocaine 1% and sedation and pain management with Versed® and fentanyl or
propofol as deemed necessary by Mansoura international hospital anesthesiologist.
Bone samples will be taken from the anterior iliac crest by the one-step electrical
drill technique (Straumann Medical, Waldenburg, Switzerland). This biopsy
technique will be done by Dr. Ibrahim El-Zayat and his colleagues. Bone
specimens will be shipped to University of Kentucky Bone histomorphometric lab.
Mineralized Bone Histology:
Iliac crest bone samples will be fixed in 100% ethanol at room temperature,
dehydrated and embedded in methylmethacrylate as previously described. Serial
sections of 3- and 7-μm thicknesses will be cut with a Microm microtome, model
HM360 (Microm, C. Zeiss, Thornwood, NY). Sections will be stained with the
modified Masson-Goldner trichrome stain and the aurin tricarboxylic acid stain to
measure the extent of aluminum deposits at the bone-osteoid interface, with
solochrome azurine stain used as control and for detection of aluminum within
bony trabecules, and with the modified Gomori trichrome stain for detection of
iron. Unstained sections will be prepared for phase contrast and fluorescent light
microscopy.

9. Bone Histomorphometry:
Cellular and dynamic parameters of bone structure, formation, and resorption will
be evaluated at standardized sites in cancellous and cortical bone using the
semiautomatic method (Osteoplan II, Kontron, Munich, Germany) at a
magnification of 200x.
The following parameters are selected for evaluation of bone turnover: (1)
activation frequency (yr-1), and (2) bone formation rate / bone surface
(mm3/cm2/yr). These parameters are obtained separately in cortical and cancellous
bone. Bone volume / tissue volume (BV/TV) (%) will be used for evaluation of
bone volume. Mineralization will be assessed by osteoid thickness (OT) and
mineralization lag time (MLT). Osteomalacia, is defined as osteoid thickness > 20
µm and mineralization lag time > 100 days. These parameters comply with the
guidelines of the nomenclature committee of the American Society of Bone and
Mineral Research. The Osteoplan II software has been programmed to transfer data
automatically to the statistical software SPSS for Windows (SPSS; Chicago IL).
Bone turnover and volume parameters will be entered as continuous variables in
statistical analyses.
Multi-slice Computed Tomography (MSCT):

10. Coronary artery calcium (CAC), thoracic aorta calcium (TAC) and abdominal
aorta calcium (AAC) scores will be assessed by using MSCT of the heart and
aorta. Non-contrast computed tomography of chest & abdomen for calcium scoring
will be performed on a Dual Source 128 Slice CT scanner (Siemens AG, Erlangen,
Germany). For CAC and TAC scores, images will be acquired from carina to the
LV apex, and the AAC images will include the distal 8 cm long segment of the
aorta ending at the aortic bifurcation as described by Multi-Ethnic Study of
Atherosclerosis (MESA) investigators. Scan parameter will be the following: ECG
gating, 64 x 0.6 collimation, 120 kVp, 80 mAs/rotation, 0.33 sec gantry rotation
time and 3 mm slice thickness. Images will be analyzed on a 3D workstation using
calcium scoring software (HeartView CT, Siemens AG, Erlangen, Germany). For
all calcium measurements (i.e., CAC, TAC, and AAC), calcification will be
identified as a plaque of ≥1 mm2 with a density of ≥130 HU and quantified using
the previously described Agatston scoring method. Image acquisition will be
performed locally in Mansoura international hospital under the supervision of the
Cardiac Radiologist. He will then read the scans and provide calcification scores to
the investigators.

11. Timeline:
1) IRB approval 2 months
2) Setups/ initiation of study/ patients recruitment 2 month
3) Run in time 12 months
4) Analysis 3 months
Statistical analysis:
Power Calculations:
Based on preliminary studies, bone formation rate/bone surface (BFR/BS) has the
highest variability among bone histomorphometric parameters (unpublished data).
We anticipate that the mean difference in BFR/BS between the two groups will be
7.4 µm3/µm2/yr (i.e., 10.8 vs. 3.4 µm3/µm2/yr). With an alpha level of 0.05 and a
sample size of 50 patients, the study will have a power of 80% to yield statistically
significant results.
Analysis of results:
Results will be expressed as mean ± SEM. All statistical tests will be two-sided.
An assigned significance level of 0.05 will be used. Normality of distribution will
be assessed by the Lilliefors test and homogeneity of variance with the Levene test.

12. Appropriate transformations of the data will be done when results do not meet
assumptions for normality.
Results will be compared between the before and after using two-way analysis of
variance (ANOVA) for pre-post measurements of BFR/BS and bone biomarkers
levels. The Kruskal Wallis test will be used to compare CACs and Aortic calcium
contents. All computations will be performed using the SPSS software package for
Windows, release 23 (SPSS, Chicago, IL).
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