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Bone Turnover in Prolonged Critical Illness: Effect of Vitamin D

Department of Intensive Care Medicine (G.V.d.B., D.V.R., P.V., P.J.W.), Pharmacy (L.D.P.) and Laboratory for Experimental Medicine and Endocrinology (R.B.), University of Leuven, B-3000 Leuven, Belgium

Address correspondence and reprint requests to: Greet Van den Berghe, M.D., Ph.D., Department of Intensive Care Medicine, University of Leuven, B-3000 Leuven, Belgium. E-mail: greta.vandenberghe{at}med kuleuven.ac.be.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
In prolonged critical illness, increased bone resorption and osteoblast dysfunction have been reported facing low 25 hydroxy vitamin D [25(OH)D] concentrations. The current study investigates the extent to which lack of nutritional vitamin D and time in intensive care contribute to bone loss in the critically ill. Prolonged critically ill patients (n = 22) were compared with matched controls and then randomized to daily vitamin D supplement of either ± 200 IU (low dose) or ± 500 IU (high dose). At intensive care admission, serum concentrations of 25(OH)D, 1,25 dihydroxyvitamin D₃, vitamin D-binding protein, ionized calcium, IL-1, and soluble IL-6-receptor were low, and PTH was normal. Circulating type-I collagen propeptides were high, alkaline phosphatase was normal, and osteocalcin was low. Bone resorption markers [(carboxy terminal cross-linked telopeptide of type I collagen (ßCTX), pyridinoline, deoxypyridinoline (DPD)] were 6-fold increased. Serum C-reactive protein (CRP) was 40-fold, IL-6 400-fold, TNF levels 5-fold, and osteoprotegerin concentrations 3-fold higher than in controls. Soluble receptor activator of nuclear factor B ligand was undetectable. High-dose vitamin D only slightly increased circulating 25 hydroxy vitamin D (P < 0.05), but 1,25 dihydroxyvitamin D₃ was unaltered. High-dose vitamin D slightly increased serum osteocalcin (P < 0.05) and decreased carboxy terminal propeptide type-I collagen (P < 0.05) but did not affect other bone turnover markers. Bone-specific alkaline phosphatase, urinary pyridinoline and DPD, and serum ßCTX markedly increased with time (P < 0.01). Circulating CRP and IL-6 decreased with time, whereas TNF and IL-1 remained unaltered. The fall in CRP and IL-6 was more pronounced with the high- than low-dose vitamin D (P < 0.05). Except for a mirroring of ßCTX rise by a fall in osteoprotegerin, cytokines were unrelated to the progressively aggravating bone resorption. In conclusion, prolonged critically ill patients were vitamin D deficient. The currently recommended vitamin D dose did not normalize vitamin D status. Furthermore, severe bone hyperresorption further aggravated (up to 15-fold the normal values) with time in intensive care and was associated with impaired osteoblast function.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
PROLONGED CRITICALLY ILL patients, defined as patients requiring vital organ function support including mechanical ventilation for more than 1 wk (1, 2, 3, 4) as well as patients treated in step-down units (5, 6) are catabolic, which is reflected by massively increased markers of bone resorption (1, 2, 4, 6, 7). Normal bone turnover depends on a tight coupling between function of mature osteoblasts and osteoclasts and requires a complex equilibrium of endocrine, immune, nutritional, and mechanical factors. The several-fold increased bone resorption in the face of impaired new bone synthesis described in prolonged critically ill patients (1, 2, 3, 4) indicates uncoupling between osteoclast and osteoblast activity, which may predispose to osteoporosis and deficient fracture healing. Prolonged critically ill patients are immobilized and have been shown to suffer from a wide variety of nutritional and endocrine disturbances. Besides immobilization, other potential causes of bone hyperresorption in this condition have been categorized as hyperparathyroidism, renal insufficiency, hypogonadism, hypercortisolism, hyposomatotropism, secondary hypothyroidism, and vitamin D deficiency.

Very low serum concentrations of 25 hydroxy vitamin D [25(OH)D] and of 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D] have been documented in prolonged critically ill patients (1, 2, 4, 6). The U.S. Academy of Science only recently increased the recommended daily allowance of vitamin D to 400 and 600 IU/d for healthy adults aged more than 50 yr and more than 70 yr, respectively (8, 9), although arguments have been raised for much higher doses (10, 11, 12). However, in line with the most recent guidelines of the American Society for Parenteral and Enteral Nutrition (13), intensive care unit (ICU) patients still receive only 200 IU of vitamin D iv when on parenteral nutrition. It has been suggested that even this low dose of vitamin D may contribute to the development of metabolic bone disease in patients on chronic parenteral nutrition (14, 15), and thus the correct dose for ICU patients remains highly controversial. In addition, it is unknown whether the alterations in vitamin D concentrations and bone turnover are present from admission onward or instead develop gradually over time in intensive care.

Few attempts have yet been made to correct bone hyperresorption in ICU patients. Combined infusion of the GH-secretagogue, GH-releasing peptide-2, together with TRH for 5 d improved the GH/IGF-I and T₃ status as well as osteocalcin production but did not affect urinary collagen cross-link excretion (2). One uncontrolled study indicated that iv pamidronate [+1,25(OH)₂D] was able to somewhat reduce bone hyperresorption in ICU patients (6). The current prospective, randomized, controlled study investigates the extent to which lack of nutritional vitamin D contributes to bone loss in surgical intensive care patients and the time course of changes in bone turnover and its regulating factors.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Study design

Patients with an anticipated ICU stay of more than 10 d were eligible for inclusion in the study. Patients younger than 18 yr, those suffering from chronic bone or kidney disease, and those who were treated with glucocorticoids before ICU admission were excluded.

On ICU admission, patients were compared with healthy age-, gender-, and body mass index-matched controls (n = 22 for blood analytes and n = 64 for urinary excretion of collagen cross-links). Patients were randomly allocated to a daily iv cholecalciferol supplement of either ±200 IU (low-dose vitamin D) or ±500 IU (high-dose vitamin D) from ICU admission onward and as long as at least some parenteral nutrition was given. Because iv supplements of vitamin D are commercially available only in the form of multivitamin supplements, the two vitamin D dose schedules were achieved by adding 10 ml Cernevit (Clinitec-Baxter, Brussels, Belgium) to the parenteral nutrition bag in the low-dose group and adding 10 ml Soluvit (Fresenius-Kabi, Puurs, Belgium) and 15 ml Vitalipid-Infant (Fresenius-Kabi) to the parenteral nutrition bag in the high-dose group, which allowed equivalent doses of other vitamins in both groups. Parenteral nutrition was prescribed by the attending physician, according to the patient’s fluid and caloric requirements. Parenteral infusion bags were prepared in laminar airflow conditions by the hospital’s pharmacy. All patients were fed enterally as soon as possible using commercially available enteral formulations that contain 200 IU vitamin D per liter. When full enteral feeding was reached, the parenteral supplements were discontinued.

Ethical aspects

The study was approved by the Institutional Review Board of the University of Leuven School of Medicine. Informed consent from a first-degree relative was obtained before patient inclusion.

Patients and concomitant treatments

Of the 33 patients included, 22 remained in intensive care for more than 10 d and were analyzed. Baseline demographic and clinical data are shown in Table 1. Twelve had been randomized to the high-dose vitamin D group and 10 to the low-dose group. The exact amount of parenteral and enteral feeding actually received by the patients was noted daily to determine the exact amount of vitamin D administered.

Other concomitant treatments included standardized, continuously administered feeding, started parenterally on ICU admission and progressively switched to combined enteral/parenteral feeding. Patients received 1847 ± 207 nonprotein kcal (20–40% administered as lipids) and 13.4 ± 2.9 g nitrogen per day.

The daily calcium intake consisted of 7.5 mg per 100 ml parenteral nutrition and 68 mg per 100 ml enteral nutrition. The daily phosphorus intake was 0–3.6 mg per 100 ml parenteral feeding and 68 mg per 100 ml enteral feeding. Daily iv supplements of 3 g magnesium sulfate were given systematically. Intensive care further comprised inotropic support with exogenous nondopaminergic catecholamines, antibiotics, analgesia, and sedation with opioids and/or benzodiazepines and intensive insulin therapy to maintain blood glucose levels between 4.4 and 6.1 mmol/liter (17). Monitoring included continuous electrocardiography, intraarterial blood pressure, central venous pressure, core and peripheral temperature, and daily fluid balance.

Blood and urine analyses

Every day at 0600 h, from ICU admission until ICU discharge or death, urine from 24-h collections and blood was sampled. Undiluted blood samples were taken through an arterial line that had been placed for clinical purposes. Serum and urine samples were frozen at -20 C and then stored at -80 C until assay.

In serum, the following concentrations were measured; 25(OH)D, 1,25(OH)₂D, vitamin D-binding protein (DBP), PTH, TNF, IL-1, IL-6, soluble IL-6 receptor (sIL-6-R), soluble receptor activator of nuclear factor-B ligand (sRANK-L), osteoprotegerin (OPG), carboxy and amino terminal propeptide of type I collagen (PICP and PINP, respectively), bone-specific alkaline phosphatase (sALP), osteocalcin (OC), and carboxy terminal cross-linked telopeptide of type I collagen (ßCTX). On a clinical basis, plasma total and ionized calcium (pH corrected), magnesium, phosphorus, urea, creatinine, and C-reactive protein (CRP) were measured daily. In those patients who were not treated with dialysis or hemofiltration (n = 16), creatinine clearance rate and urinary excretion of calcium, phosphorus, creatinine, DBP, and the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) was also measured.

Assays

Serum CRP concentrations were measured by immunoturbidimetric assay (Roche-Itachi-Modular-P, Roche, Basel, Switzerland). Serum concentrations of TNF, IL-1, and IL-6 were measured by a solid-phase enzyme amplified sensitivity immunoassay (BioSource Europe SA, Nivelles, Belgium). Within-assay and between-assay coefficient of variation (CV) were less than 5% and 8%, respectively, for all assays. Serum sIL-6R was measured by ELISA (BioSource) with a within- and between-assay CV of 5.4% and 7.3%, respectively.

Serum OPG levels were measured by enzyme immunoassay (BI-20402, Biomedica Gruppe, Vienna, Austria). The detection limit was 2.7 pg/ml. The between- and within-assay CVs were less than 10%. Serum sRANK-L levels were measured by enzyme immunoassay (BI-20422, Biomedica). The detection limit was 0.4 pmol/liter. The between- and within-assay CVs ranged between 5% and 9%.

Serum intact PTH was measured by a two-site immunometric assay that detects only PTH(1–84) but not the large PTH(7–84) fragments (18, 19). Serum 25(OH)D was measured by competitive binding assay, 1,25(OH)₂D by RIA, and DBP by radial immunodiffusion, as previously reported (20).

Serum levels of PICP and PINP were measured by RIA (Orion Diagnostica, Espoo, Finland). The within- and between-assay CV was 3% and 4% (PICP) and 5.1% and 8.8% (PINP), respectively. Serum sALP was determined by immunoradiometric assay (Tandem-R Ostase, Hybritech Inc., San Diego, CA). The within- and between-assay CV was 7% and 8%. Serum OC was measured by a homologous human osteocalcin RIA, as described (1, 2, 4, 21). The within- and between-assay CVs were 5% and 7%, respectively.

Serum ß-CTX levels were measured by electrochemiluminescence immunoassay (Roche Elecsys Systems, Mannheim, Germany) with a between- and within-assay CV of 1.8% and 4.3%. Urinary pyridinolines (PYD and DPD) were measured in 24-h urine collections, as previously described (1, 2, 4). Values are expressed as nanomoles per millimoles creatinine. Urinary creatinine was measured colorimetrically. Interassay CVs were 11.5% and 13.3% for PYD and DPD, respectively (n = 12) and within-assay CVs were 10.2% and 12.5% (n = 9).

Statistical analysis

Data are presented as means ± SEM for precision and means ± SD or medians (25th to 75th percentile) for distribution. Between-group comparisons were done using ANOVA with post hoc Fisher’s protected least significant difference for multiple comparisons, t test, Mann-Whitney U test (with Bonferroni correction in case of multiple comparisons), and ² test, when appropriate. The time effect was analyzed by repeated-measures ANOVA or paired t tests or Wilcoxon signed rank test, with Bonferroni correction for multiple testing when appropriate. Pearson product-moment correlation coefficients (R) and determination coefficients (R²) were calculated for quantifying the relation between variables. Two-sided P < 0.05 were considered significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Description of the studied variables at ICU admission

On ICU admission, critically ill patients presented with elevated plasma concentrations of urea, creatinine, and CRP (Table 1).

Plasma level of total calcium was normal and whole blood ionized Ca ²⁺ was low, the latter not related to CRP concentration or the reason for ICU admission. Plasma phosphorus level was normal, and plasma magnesium slightly increased probably related to its iv supplementation. Serum PTH level was not significantly different from controls, but high as well as low levels of PTH occurred, high levels being linked to the presence of hypocalcemia (ionized calcium <1.1 mmol/liter) in 36% of patients. PTH indeed correlated inversely with Ca²⁺ on admission to ICU (R = -0.48, R² = 0.23, P = 0.03).

Serum levels of 25(OH)D and 1,25(OH)₂D were less than half of those in controls. This was in part related to the reason for ICU admission, with significantly higher 25(OH)D and 1,25(OH)₂D levels in the neurotrauma/polytrauma group as compared with the abdominal surgery and cardiothoracic surgery group. This relation was partly explained by the younger age of the trauma patients (33 ± 5 yr vs. 56 ± 10 yr and 67 ± 3 yr, respectively, P = 0.003). There was a negative correlation between serum 1,25(OH)₂D levels and serum TNF levels (R = -0.51, R² = 0.26, P = 0.02) and a positive correlation with creatinine clearance in the patients who were not on dialysis (R = +0.89, R² = 0.38, P = 0.003). Stepwise regression analysis revealed creatinine clearance, but not TNF, as an independent determinant of 1,25(OH)₂D. Serum DBP concentrations were reduced, equally in both groups, to 72% of normal. The molar ratio of 1,25(OH)₂D over DBP, an estimate of the free 1,25(OH)₂D concentration (20) was also reduced by 50%, compared with controls. Survivors and nonsurvivors were not distinguishable by vitamin D status at baseline.

Serum PICP concentrations were 7.6-fold, but PINP values, positively correlated with PICP (R = +0.57, R² = 0.33, P = 0.006), were only 1.7-fold increased; sALP was normal and OC concentration was only 63% of normal. These changes were independent of the reason for ICU admission or severity of illness and unrelated to age or outcome.

Urinary markers of bone resorption, DPD and PYD, were 6- and 5-fold increased, respectively, in line with 6-fold elevated serum levels of ßCTX. These changes were independent of type and severity of illness and unrelated to age or outcome. Serum ßCTX concentrations were not different in patients with or without acute renal failure requiring dialysis or hemofiltration. The DPD/PYD ratio was slightly but significantly lower in patients than controls.

Urinary excretion of calcium and phosphorus on ICU admission was low. On admission, mild microalbuminuria (>30–164 mg/24 h) was present in 53% of patients who did not require dialysis. Except in two patients, no detectable amounts of DBP were found in urine.

Serum levels of IL-1 and sIL-6-R were somewhat lower than in controls, whereas serum IL-6 was more than 400-fold and TNF levels 5-fold increased. Serum OPG concentration was 3-fold elevated, whereas sRANK-L was undetectable in the majority of the patients and controls. Cytokine levels on admission were independent of bone turnover markers except for a positive correlation between serum IL-6 and sALP (R = +0.58, R² = 0.34, P = 0.006) and a trend for a positive correlation between ßCTX and TNF (R = +0.37, R² = 0.14, P = 0.1). On admission, IL-6 was the only cytokine that predicted mortality [1809 (987–2149) pg/ml in nonsurvivors vs. 324 (139–512) pg/ml in survivors, P = 0.04].

Vitamin D supplementation

The mean daily iv vitamin D dose actually given in the low-dose group was 134 ± 13 IU vs. 404 ± 43 IU in the high-dose group (Fig. 1). The total daily vitamin D doses in the two groups remained significantly different during the first 10 d (225 ± 20 IU vs. 491 ± 31 IU; P = 0.0001) but no longer thereafter (Fig. 1) because of increasing enteral feeding with equivalent formulae in both groups.

View larger version (44K): [in this window] [in a new window]   FIG. 1. Vitamin D intake in the two study groups. Randomized iv dose of vitamin D, vitamin D intake with increasing amount of tube feeding, and total intake of vitamin D (daily units of cholecalciferol, means ± SEM) in the two study groups during the first 10 d and the last day in ICU. Open bars represent the low-dose group (n = 10), and hatched bars represent the high-dose (n = 12) vitamin D group.

Serum concentrations of 25(OH)D in the high-dose vitamin D group were higher than in the low-dose group only on d 2, d 6, and d 7 (P = 0.05) (Fig. 2), but at no time did they reach normal levels. The serum concentrations of 25(OH)D did not correlate with the amount of vitamin D given at any time point but were lower in the nonsurvivors, compared with survivors (8.1 ± 1.3 µg/liter vs. 15.2 ± 5.0 µg/liter, P = 0.009) on the last day of intensive care. The presence of acute renal failure did not affect serum 25(OH)D levels.

View larger version (85K): [in this window] [in a new window]   FIG. 2. Effect of 200 IU and 500 IU of vitamin D supplement on vitamin D metabolism. Serum concentrations of 25(OH)D, 1,25(OH)2D, DBP, and the molar ratio of 1,25(OH)2D over DBP in the two study groups (left panels; open bars represent the low vitamin D dose, and the hatched bars represent the high vitamin D dose) as well as in patients with and without acute renal failure (right panel; dark bars represent patients on dialysis or hemofiltration, light bars represent patients not requiring dialysis or hemofiltration) Data are means ± SEM. *, P < 0.05

Serum DBP levels, low on admission, were not affected by a higher vitamin D supplement but increased gradually with time (P = 0.0001) in both groups, reaching normal levels after 10 d of intensive care. The time-dependent increase in serum DBP was absent in the three nonsurvivors.

The free 1,25(OH)₂D index was also unaffected by vitamin D supplementation. After 1 wk of intensive care, the molar ratio of 1,25(OH)₂D over DBP was lower in the patients who developed acute renal failure requiring dialysis or hemofiltration than in those who did not require this treatment. Nonsurvivors tended to have lower molar ratios of 1,25(OH)₂D index than survivors.

Effect on calcium and PTH

Total and ionized calcium, phosphorus and PTH concentration, and urinary calcium and phosphorus excretion did not change significantly over time in either of the vitamin D treatment groups. Patients who developed acute renal failure requiring continuous venovenous hemofiltration or dialysis had higher ionized calcium and lower serum PTH levels than patients without acute renal failure from d 4 until d 9 (P 0.05).

Effects on markers of bone turnover

Osteoblast function The elevated PICP level on ICU admission increased from d 1 to d 7 in the low-dose vitamin D group with no significant change in the high-dose group, whereas PICP decreased significantly in the high-dose group from d 8 onward (P 0.03) (Fig. 3). Likewise, serum PINP levels tended to increase from admission to d 10 (P = 0.07) in the low-dose group with no significant change in the high-dose group. On d 10, PINP remained positively correlated with PICP (R² = 0.79, p < 0.0001). Serum levels of sALP were not influenced by vitamin D treatment but increased gradually with time in ICU reaching 3-fold higher levels on the last day of intensive care (P = 0.0001). Serum OC concentrations, already low at baseline, decreased further in the low-dose vitamin D group but remained at the baseline level in the high-dose group (P 0.03) (Fig. 3). Serum OPG was 3-fold increased upon ICU admission but decreased with time (P = 0.006, Fig. 4) independently of vitamin D supplementation. On the last day of intensive care, serum PICP (P = 0.0001) and OPG (P < 0.0001) levels were several-fold higher in nonsurvivors as compared with survivors.

View larger version (49K): [in this window] [in a new window]   FIG. 3. Effect of 200 IU and 500 IU of vitamin D supplement on markers of bone formation. Serum concentrations of PICP, sALP, and OC during the first 10 d and the last day in ICU. Open bars represent the low-dose vitamin D group (n = 10), and hatched bars represent the high-dose group (n = 12). Data are means ± SEM. The elevated PICP level on ICU admission increased from d 1 to d 7 in the low-dose vitamin D group with no significant change in the high-dose group, whereas PICP decreased significantly in the high-dose group from d 8 onward (P 0.03). Serum levels of sALP were not influenced by vitamin D treatment but increased gradually with time in ICU, reaching 3-fold higher levels on the last day of intensive care (P < 0.0001). Serum OC concentrations, already low at baseline, decreased further in the low-dose vitamin D group but remained at the baseline level in the high-dose group (P 0.03).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Aggravation of bone hyperresorption with time in ICU. A time-dependent increase in serum ßCTX (all patients) and urine DPD (normalized for urine creatinine in those patients who did not require dialysis or hemofiltration) levels, both already elevated on ICU admission, was observed. This was mirrored by a time-dependent decrease in serum OPG levels (all patients). Data are means ± SEM. The horizontal gray area represents the normal range (means ± 2 SD).

Urinary PYD and DPD excretion, already several-fold elevated on admission to intensive care, further increased 2.5-fold with time in ICU (Fig. 4), similarly in both study groups. A similar time-dependent increase in serum ßCTX levels was observed. In all sampling points for which both a serum and urine level of collagen cross-links was available, a positive correlation was observed between serum ßCTX and urinary pyridinoline (R = +0.41, R² = 0.17, P = 0.0001) and DPD (R = +0.56, R² = 0.31, P = 0.0001). On the last day of intensive care, serum ßCTX levels were 3-fold higher in nonsurvivors as compared with survivors (P = 0.04).

Osteoblast-osteoclast coupling

Neither of the osteoblast function markers was correlated to markers of osteoclast function at any time during ICU stay. Analyzing all samples together in which both serum ßCTX and OC was measured, serum ßCTX level did correlate positively with serum OC (R = +0.44, R² = 0.19, P = 0.0001), albeit weakly and with a reduced slope (CTX = 0.18 + 0.04 OC).

Effect on inflammation

Elevated CRP and IL-6 levels, observed on ICU admission, decreased significantly with time in ICU in both study groups (Fig. 5), whereas levels of TNF and IL-1 remained unaltered (Fig. 6). The fall in CRP was significantly more pronounced in the high-dose vitamin D group, compared with the low-dose group between d 3 and d 7 (Fig. 5); likewise in the high-dose vitamin D group, IL-6 levels decreased from admission to d 4 (P = 0.03), whereas they remained unaltered in the low-dose group (Fig. 5). This antiinflammatory effect was independent of the reason for ICU admission and the severity of illness.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Antiinflammatory effects of vitamin D in the critically ill. Plasma concentrations of CRP and IL-6 serum concentrations over time in ICU are depicted for the two study groups. Open bars represent the low-dose vitamin D group (n = 10), and hatched bars represent the high-dose group (n = 12). Data are means ± SEM. Elevated CRP and IL-6 levels, observed on ICU admission, decreased significantly with time in ICU in both study groups. The fall in CRP was significantly more pronounced in the high-dose vitamin D group, compared with the low-dose group between d 3 and d 7; likewise in the high-dose group, IL-6 levels decreased from admission to d 4, whereas they remained unaltered in the low-dose group. *, P < 0.05.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Circulating cytokine levels with time in ICU. Elevated serum IL-6 levels, observed on ICU admission, decreased significantly with time in ICU, whereas elevated levels of TNF and low IL-1 remained unaltered. Serum sIL-6-R levels were low on ICU admission but normalized with time in ICU. Data are means ± SEM (P < 0.01 for the comparison on d 0 and last day for IL-6 and sIL-6-R. The horizontal gray area represents the normal range (means ± 2 SD).

Correlation of time course of bone turnover with evolution of cytokines

Except a mirroring of the increase in CTX by the decrease in OPG (Fig. 4), the time course of cytokines was not related to the progressively aggravating bone resorption with time in ICU.

Effect of concomitant medication

The concomitant treatment with glucocorticoids, thyroid hormones, or any other ICU medication did not significantly affect vitamin D status, calcium, PTH, markers of bone turnover, or inflammation at any of the studied time points.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Prolonged critically ill patients were vitamin D deficient throughout time in ICU. This deficiency was not corrected by a more than doubling of vitamin D intake. Long-stay intensive care patients also revealed pronounced bone hyperresorption, despite low urinary calcium excretion, as part of the catabolic state. In contrast, bone formation markers suggested that, although immature osteoblasts appear highly active as reflected by PICP and PINP levels, a maturation defect underlies low activity of mature osteoblasts, as indicated by sALP and OC concentrations. This constellation resembles the abnormal matrix formation of patients with Paget’s disease. Local effects of cytokines on the RANK-RANK-L system, insufficiently compensated by OPG, may be involved. Whether this abnormal bone turnover during critical illness contributes to slow healing of fractures and surgical bone lesions remains to be investigated.

Mild vitamin D deficiency is frequent in Western European populations and is more severe in the elderly (8, 22, 23). Prolonged critically ill patients were severely vitamin D deficient with mean values of 11 ng/ml and even lower in elderly patients. The contribution of preexisting diseases on the severity of vitamin D deficiency cannot be derived from the present study. The iv vitamin D replacement dose of about 200 IU/d used in most ICUs, in line with updated guidelines for parenteral nutrition (13), reflects the old internationally recommended adequate intake. Only very recently the latter has been increased to 400 for adults (50–70 yr) and 600 IU/d for elderly subjects (>70 yr) (8, 9). The present prospective, randomized study clearly indicates that neither the old nor the newly recommended dose was able to correct vitamin D deficiency in prolonged critically ill patients. In addition, intensive care patients clearly revealed decreased total and free 1,25(OH)₂D levels, which cannot be explained by simple precursor deficiency (8, 24). There was no significant improvement by more than doubling of the vitamin D supplement. Thus, impaired kidney function appears a major factor (negative correlation between serum 1,25(OH)₂D and creatinine clearance) but cytokine release (negative correlation between serum 1,25(OH)₂D and TNF) and perhaps also GH/IGF-I deficiency (1, 4) may have aggravated the impaired 1-hydroxylase activity. The effect of higher replacement doses, such as those advised for noncritically ill adults by some groups, (10, 11, 12), should therefore be explored.

DBP is decreased in intensive care patients and nonrecovery of the low levels of this liver-derived protein appears a predictor of ICU mortality (25). Actin, released by organ trauma or dysfunction, binds to DBP (26). This complex formation avoids fibrillar actin formation and accelerates DBP and 25(OH)D clearance (27). However, the modestly decreased DBP level cannot explain the extremely low 1,25(OH)₂D concentration because the 1,25(OH)₂D:DBP ratio, an index of free 1,25(OH)₂D (20), is also decreased.

Surprisingly, most intensive care patients do not reveal secondary hyperparathyroidism, which would be expected on the basis of mild hypocalcemia, severe 1,25(OH)₂D deficiency, and renal insufficiency. The cause of this inappropriate PTH release remains hitherto unclear.

Prolonged critically ill patients revealed a dramatically increased bone resorption as indicated by the 6-fold elevated ßCTX levels in serum and an equally elevated urinary excretion of collagen cross-links. Moreover, bone resorption more than doubled once again during ICU stay (28, 29, 30). RANK-RANK-L is the common final pathway for osteoclast activation and can be activated by several receptors. The vitamin D and PTH receptor pathways are potent stimulators of the RANK-RANK-L system but are unlikely to be involved here because their ligands, 1,25(OH)₂D and PTH, are not increased. However, to exclude a role for vitamin D, a full normalization of the vitamin D status would be required. Circulating levels of inflammatory cytokines, such as TNF and particularly IL-6, were several-fold elevated upon intensive care admission and only gradually and moderately decreased, if at all, during the further observation period (31). Both cytokines are known to be potent and synergistic activators of RANK-RANK-L (32). The critical role of IL-6 in osteoclastogenesis is well documented (33, 34, 35, 36). Furthermore, excess IL-6 contributes to increased bone turnover in Paget’s disease (37).

Surprisingly, however, markers of bone resorption in the critically ill patients did not significantly correlate with serum IL-6, IL-1, or TNF concentrations. This suggests that other mechanisms may have been active or, alternatively, that circulating cytokines do not adequately reflect local cytokine production and RANK-RANK-L activation. Bone resorption increased with time in ICU despite decreasing inflammation as reflected by CRP levels and cytokine concentrations. This paradox can be explained by the time required for osteoclasts to fully mature after recruitment and an increased life span of osteoclasts. Indeed, most of the measured cytokines, as well as those we did not measure (e.g. IL-11, prostaglandins, leukemia inhibitory factor, and chemokines), not only stimulate osteoclastogenesis but also prolong their half-life through inhibition of apoptosis (38). Moreover, circulating levels of the decoy receptor for RANK-L, OPG, decreased with time in ICU, which may have allowed the local RANK-L to gain activity. Furthermore, normalizing sIL-6-R concentration with time may have enhanced local IL-6 activity because IL-6 activates the gp130 receptor of osteoclast precursor cells only in the presence of the sIL- 6-R (39).

Whether osteoclast hyperactivity in the critically ill is unequivocally due to stimulation of RANK-RANK-L or achieved by a direct TNF (39) or a synergistic RANK-L-TNF effect on osteoclast precursors (40) cannot be firmly established by a clinical study, unless bone or bone marrow would be available. Low sRANK-L concentration measured in serum, reflecting either true low RANK-L signal or an artifact because of low assay sensitivity, does not exclude high local expression of RANK-L in bone environment. High serum concentrations of the decoy receptor OPG, however, indirectly points to a role of the RANK-RANKL system. Interleukin-6 is known to stimulate both RANK-L and OPG in osteoblasts, but RANK-L activation exceeds the inhibitory effects of the generated OPG.

Bone formation markers at admission to intensive care were clearly abnormal because PICP was frankly elevated (7-fold) and PINP mildly increased (1.7-fold), whereas sALP levels were normal and OC was low. These osteoblast proteins are developmentally regulated, with collagen synthesis preceding sALP expression and OC production being the final marker of fully mature osteoblasts (41, 42). Therefore, the constellation of abnormalities in circulating levels of osteoblast markers suggest a large excess of immature osteoblasts, which secrete predominantly collagen and alkaline phosphatase but fewer than normal mature osteoblasts. The discrepancy between PICP and PINP can be explained by the slower degradation of PICP in severe inflammation because of down-regulation of the mannose 6-phophate or IGF-II receptor (35). Therefore, circulating PICP levels during critical illness may somewhat overestimate procollagen synthesis. Full maturation of osteoblasts may be hampered by inflammation and the hormonal abnormalities in ICU patients (1, 2, 3, 4), explaining the persistently low OC levels (Fig. 3).

Overall, bone resorption markers are far more elevated than markers of bone formation during critical illness. Whereas the studied resorption markers probably faithfully reflect the degree of bone loss, the observed alterations in markers of osteoblast function do not allow a precise estimation of true bone formation. The large discrepancy between high calcium efflux from bone and low urinary calcium excretion, without other obvious calcium loss, suggests that bone matrix formation and calcium deposition are increased. Calcium kinetic studies or similar procedures will be needed to clarify the complex handling of calcium in prolonged critically ill patients.

Bone loss in metabolic bone diseases is rarely increased to the same extent as in the studied ICU patients. Indeed, in menopause and other types of hypogonadism, primary hyperparathyroidism (27, 43), hyperthyroidism (44), or AIDS (45), a more than doubling of bone resorption is exceptional. Local bone turnover may be markedly increased in rheumatoid arthritis or Paget’s disease but the total body bone loss rarely matches the level observed during critical illness. There are several factors that inferentially contribute to altered bone turnover of ICU patients. Immobilization by itself is probably a minor contributor because a 10-d bed rest in healthy subjects does not significantly (46), or only mildly (47, 48, 49), increase bone resorption markers. Hormonal alterations in prolonged severe illness comprise impaired GH/IGF-I secretion and a low T₃ syndrome, both of which would predict low instead of high bone turnover. Hypogonadism and mild hypercortisolism are also present and could accelerate bone turnover, although effects of such endocrinopathies are modest and have a much slower onset. PTH was normal and (free) 1,25(OH)₂D levels were frankly decreased, and therefore calciotropic hormones also cannot be held responsible. Intensive care patients therefore represent a unique model of high bone turnover (6- to 15-fold increased bone resorption), and further studies may reveal interesting aspects of rapid activation of bone cells and mechanism of coupling and uncoupling of resorption with formation.

The antiinflammatory effects of exogenous vitamin D, as revealed by suppression of serum CRP and IL-6 concentrations as long as circulating 25(OH)D levels were significantly increased, are fascinating, particularly in view of the potential role of excessive inflammation in causing multiple organ failure. 1,25(OH)₂D is known to suppress inflammatory cytokines, especially IL-6, in several cell types (50) (Segaert, S., and R. Bouillon, unpublished observations) and has a favorable effect on monocyte function (51). Animal studies have indeed confirmed strong immune modulating and antiinflammatory effect of 1,25(OH)₂D and its less calcemic analogs (51, 52, 53). Because serum 1,25(OH)₂D concentrations were not increased by the treatment, local 1-hydroxylase activity in immune cells may have been the mediator (51). Whether rapid and full vitamin D replacement therapy would further enhance such favorable effects requires more study.

What are the implications of these observations? There is no doubt that the primary concern of intensive care is to support vital organ function to decrease mortality and morbidity. However, studies on bone degradation may provide novel insights in the mechanisms involved in the therapy-resistant catabolic state of such patients. Moreover, the accelerated bone resorption and the peculiar osteoblast dysfunction may contribute to slow healing of traumatic and surgical bone lesions. Finally, the intensive care patient appears an excellent clinical model for evaluation of pathogenic mechanisms resulting in massively increased bone turnover. The recently developed animal model of prolonged critical illness will allow study of these mechanisms in greater detail (54). Finally, the antiinflammatory effect of vitamin D opens new therapeutic perspectives for improving outcome.

In conclusion, critically ill patients requiring prolonged intensive care are vitamin D deficient. Neither the old nor the newly recommended daily supplementation can normalize 25(OH)D or 1,25(OH)₂D levels. Additional data on the effects of rapid and full normalization of the vitamin D status on bone turnover and inflammation are needed to redefine the recommended daily allowance of vitamin D in ICU patients. Furthermore, severe bone hyperresorption is present in prolonged critically ill patients, despite low urinary calcium excretion, whereas markers of bone formation indicate activated but immature osteoblasts. The increased bone turnover is conceivably related to massively increased cytokine production and further aggravates with time in ICU. The prolonged critically ill patient is therefore a unique model of high bone turnover.

	Acknowledgments

 
We thank Willy Coopmans, Erik Van Herck, Suzanne Marcelis, Ivo Jans, and Ilse Milants for expert technical support; the medical and nursing staff of the intensive care unit for patient care; and Bernadette Minten and Jenny Gielens for secretarial assistance.

	Footnotes

 
This work was supported by grants from the Belgian Research Foundation F.W.O.-Vlaanderen [G.0144.00 and G.3C05.95N (to G.V.d.B.) and G.0241.01 (to R.B.)] and the University of Leuven (OT/99/32 to G.V.d.B.).

This work was presented in part at the 15th Annual Congress of the European Society of Intensive Care Medicine, September 29 to October 2, 2002, Barcelona, Spain.

Abbreviations: CRP, C-reactive protein; ßCTX, carboxy terminal cross-linked telopeptide of type I collagen; CV, coefficient of variation; DBP, vitamin D-binding protein; DPD, deoxypyridinoline; ICU, intensive care unit; OC, osteocalcin; 1,25(OH)₂D, 1,25 dihydroxyvitamin D₃; 25(OH)D, 25 hydroxy vitamin D; OPG, osteoprotegerin; PICP, carboxy terminal propeptide of type I collagen; PINP, amino terminal propeptide of type I collagen; PYD, pyridinoline; sALP, bone-specific alkaline phosphatase; sIL-6-R, soluble IL-6 receptor; sRANK-L, soluble receptor activator of nuclear factor-B ligand.

Received February 28, 2003.

Accepted June 24, 2003.

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