• Open Access

Calcium Regulating Hormones and Serum Calcium and Magnesium Concentrations in Septic and Critically Ill Foals and their Association with Survival

Authors


  • Previously presented in abstract form at the American College of Veterinary Internal Medicine Forum 2007, June 5–9.

Corresponding author: R.E. Toribio, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210; e-mail: toribio.1@osu.edu.

Abstract

Background: Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival.

Hypothesis: Blood concentrations of ionized calcium (Ca2+) and magnesium (Mg2+) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival.

Animals: Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of ≤7 days were included.

Methods: Prospective, observational study. Blood was collected at initial examination for analysis. Foals with positive blood culture or sepsis score ≥14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays.

Results: Septic foals had decreased Ca2+ (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg2+, PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82).

Conclusions and Clinical Importance: Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.

Disorders of calcium homeostasis are frequently documented in critically ill humans and animals and have been associated with the severity of illness.1,2 Hypocalcemia, hypomagnesemia, and parathyroid gland dysfunction have been reported in septic and critically ill adult horses with endotoxemia and gastrointestinal disease,2–4 but not in foals. Sepsis in neonates is cited as the leading cause of death in critically ill foals,5–8 with survival rates of 10 to 70%.5,7,8 Several studies of foals have investigated the prognostic value of clinicopathologic variables for survival,6,9–13 but few have evaluated the association between endocrine factors and rate of death in hospitals.8,14 There is limited information regarding calcium and magnesium regulation in healthy and diseased foals.

Studies in healthy horses (and foals) have revealed that foals have higher serum Mg2+ and total magnesium (TMg) concentrations and lower Ca2+ and TCa concentrations than adult horses.15 Dietary intake of magnesium is also considered the major determinant of serum magnesium concentrations in horses,16 and unlike calcium homeostasis, Mg2+ is not believed to be under specific endocrine homeostatic control.

Abbreviations
ANOVA

analysis of variance

Ca2+

ionized calcium

95% CI

95% confidence interval

CT

calcitonin

1,25-D3,

1,25-dihydroxyvitamin D3

IgG

immunoglobulin G concentration

IRMA

immunoradiometric assay

Mg2+

ionized magnesium

OR

crude odds ratio

P

phosphorus

PCT

procalcitonin

PTH

parathyroid hormone

PTHrP

parathyroid hormone-related peptide/protein

RIA

radioimmunoassay

TCa

total calcium

TMg

total magnesium

Regarding endocrine variables, limited information exists in critically ill horses. Parathyroid hormone (PTH) has important homeostatic actions in horses that increase extracellular Ca2+ in response to hypocalcemia by increasing renal reabsorption and bone resorption of calcium or increasing the renal fractional excretion of inorganic phosphorus during hyperphosphatemia. Parathyroid-related peptide (PTHrP) is important for the regulation of fetal calcium concentrations and transplacental calcium transport.17 In adult horses, increased PTHrP concentrations are associated with humoral hypercalcemia of malignancy.18,19

Both increases and decreases in PTH concentrations have been documented in critically ill horses and humans with and without ionized hypocalcemia.2,3,20 Hypomagnesemia also occurs in septic humans and horses where decreases in Mg2+ concentrations interferes with the parathyroid gland response to hypocalcemia.3,4,21

The purpose of this study was to determine the blood concentrations of Ca2+, Mg2+, TCa, and calcium regulating hormones (PTH, PTHrP, and calcitonin [CT]) in septic foals, sick nonseptic foals, and healthy foals and to determine their association with survival. We hypothesize that endocrine dysregulation of calcium homeostasis exists in septic foals.

Materials and Methods

Animals

All full-term foals of ≤7 days old of both sexes and any breed admitted to The Ohio State University Veterinary Teaching Hospital (OSU) and Hagyard Equine Medicine Institute (HEMI) were studied. Data were collected on foals from 2 consecutive northern hemisphere Thoroughbred foaling seasons (season 1 and season 2). Foals were then classified into 1 of 2 groups: septic and sick nonseptic. Foals in the septic group were defined as having a positive blood culture or a sepsis score ≥14.13 Sick nonseptic foals were those foals that presented for conditions other than septicemia including meconium impaction, hypoxic ischemic encephalopathy, various orthopedic conditions, and complete or partial failure of transfer of passive immunity. These foals also had negative blood cultures and sepsis scores ≤10. A 3rd group of healthy foals from various breeding farms that were <72 hours old at time of examination were used as healthy controls. They were determined to be healthy based on physical examination, a normal CBC, serum biochemistry, serum immunoglobulin G (IgG) concentration >800 mg/dL, and sepsis scores ≤5. Foals that had received prior isotonic crystalloid fluids, antimicrobials, or oral immunoglobulin supplementation (banked colostrum) were included for analysis. No foals that had received exogenous calcium or magnesium before sample collection.

Survival was defined as survival to discharge from the hospital. Nonsurvival was defined as death from progressively worsening disease or euthanasia based on a grave medical prognosis. Foals that were electively euthanized for reasons related to owner financial limitations or personal decisions to not proceed with treatment were not included in the study to avoid bias.

This study was approved by The Ohio State University Veterinary Teaching Hospital executive committee and adheres to the principles for the humane treatment of animals in veterinary clinical investigations as stated by the American College of Veterinary Internal Medicine and National Institute of Health guidelines. Owner consent was obtained before inclusion in the study.

Data Collection

A history including expected foaling date, maternal health during pregnancy, and administered medications was obtained. Categorical variables assessed included age at presentation, breed, and sex. Physical examination findings including presence of a septic focus that included arthropathy, pneumonia, diarrhea, uveitis, or omphalitis were recorded. Variables analyzed and compared among all foals included clinical heart rate, respiratory rate, temperature, mucous membrane color, capillary refill time, presence of cold extremities, peripheral pulse quality, mental status, thoracic and abdominal auscultation, ocular examination and umbilical examination, and calculated sepsis score.

Blood variables assessed in all foals included a CBC,a serum biochemistry,b blood l-lactate concentration,c plasma fibrinogen concentration,d serum IgGb concentration, blood culture results, and hormone concentrations (serum PTH, serum PTHrP, and plasma CT concentrations). PTH was the only hormone measured in foals from foaling season 2 as only PTH concentration was significantly (P < .05) associated with survival in foals from season 1. Serum Ca2+ and Mg2+ concentrations were determined with ion-selective electrodes.e For foals from season 1, serum Ca2+ and Mg2+ were measured on stored serum samples at the end of the 6-month sample collection period. For foals from season 2, a separate aliquot of serum taken at the time of sampling was stored under anaerobic conditions in sealed 1 mL syringes for Ca2+ and Mg2+ determinations.

Sampling

Whole blood from hospitalized foals was obtained by jugular venepuncture within 1 hour from the time of admission before any medical therapy and placed into plain serum clot tubes and chilled aprotinin-EDTA tubes. Aprotinin was added to preserve sample integrity (500 kU/mL whole blood in an EDTA blood tube). A total of 20 mL of venous blood were obtained for hormone assays, stored in ice water and centrifuged within 12 hours at 5 °C, 2,000 g for 15 minutes. Serum and plasma were then aliquoted and stored at −80 °C until analyzed. Blood samples from healthy foals were collected during a routine newborn foal physical examination at the farm. These foals were <72 hours of age.

Hormonal Assays

A solid phase, double antibody commercial immunoradiometric assayf (IRMA) was utilized to determine serum PTH concentrations in foals from season 1 and season 2. Two separate assays kits were used, 1 for foals from season 1 and the other for foals from season 2. Plasma PTHrP concentrations were measured in foals from season 1 with a human-specific IRMA.g The PTH and PTHrP immunoassays have been validated in horses.19,22 Plasma CT concentrations were determined in season 1 foals by an ultrasensitive direct radioimmunoassayh that has been validated in our laboratory. The intra and interassay coefficients of variation for this assay were determined with samples from adult horses. Intra-assay coefficients of variation for low (11.1 ± 5.5 pg/mL), moderate (39.7 ± 4.7 pg/mL), and high (114.4 ± 4.3 pg/mL) CT concentrations were 12, 11.9, and 3.8%, respectively. The interassay coefficients of variation the same samples were 11.4, 6.4, and 10.0%, respectively. The sensitivity of the CT assay was 0.7 pg/mL.

Statistical Analyses

Data were summarized by calculating descriptive statistics.i,j,k Frequency distributions of categorical variables were evaluated, and means, medians, standard errors of the mean, and ranges were calculated for continuous variables. Continuous variables were further categorized to facilitate analysis and determine crude odds ratios (OR) and 95% confidence intervals (CI). The dependent variable was survival. All data were assessed for normality by the Shapiro-Wilk statistic. Parametric and nonparametric testing was performed depending on the distribution of the data. Comparisons between all groups were assessed by Kruskal-Wallis 1-way ANOVA and multiple comparisons were performed with the Dunn posthoc test when statistically significant (P < .05). Comparisons between survivors and nonsurvivors within each group of foals were assessed by Mann-Whitney U statistic. Correlation between individual variables was assessed by the Spearman coefficient (r). Significance was set a P < .05. Values are recorded as median and range, unless otherwise stipulated.

Results

Study Populations

A total of 146 foals from both foaling seasons fulfilled the criteria for inclusion (Tables 1 and 2). There were 83 foals in season 1 (HEMI = 47 foals; OSU = 36 foals), where 47/83 (57%) were classified as septic, 24/83 (29%) were classified as sick nonseptic, and 12/83 (14%) healthy foals were assessed. There were 63 foals (HEMI = 56 foals; OSU = 7 foals) in season 2, where 35/63 (56%) were septic, 16/63 (25%) were sick nonseptic, and 12/63 (19%) were considered healthy. The median age for all foals in season 1 was 24 hours (range: septic, 1–168 hours; sick nonseptic, 1–144 hours; healthy, 24–72 hours) and 12 hours in season 2 (range: septic, 1–72 hours; sick nonseptic, 1–24 hours; healthy, 24–72 hours). The median age for all foals (both seasons) was 18 hours.

Table 1.   Breed and sex characteristics of foals included in 2 foaling seasons (n = 146; values are expressed as a fraction of the total number of foals and percentage [%]).
VariableTotal NumberSepticSick NonsepticHealthy
Breed
 Thoroughbred103/146 (71%)54/82 (66%)26/40 (65%)23/24 (96%)
 Non-Thoroughbred43/146 (29%)28/82 (34%)14/40 (35%)1/24 (4%)
Sex
 Colt89/146 (61%)50/82 (61%)25/40 (62%)14/24 (58%)
 Filly57/146 (39%)32/82 (39%)15/40 (38%)10/24 (42%)
Table 2.   Overall survival rates of 146 neonatal foals from 2 foaling seasons (values expressed as a fraction of the total number of foals who survived and percentage [%]).
 Total
Number
SepticSick
Nonseptic
Healthy
Season 156/83 (67%)23/47 (49%)21/24 (87.5%)12/12 (100%)
Season 242/63 (67%)17/35 (49%)13/16 (81%)12/12 (100%)
Both seasons98/146 (67%)40/82 (49%)34/40 (85%)24/24 (100%)

Breeds represented in the total foal population included Thoroughbred (n = 103) and non-Thoroughbred breeds (n = 43) including Standardbred (n = 21), Quarterhorse (n = 13), Saddlebred (n = 2), American Paint Horse (n = 2), and 1 each of the following Hanoverian, Dutch Warmblood, Friesian, Appaloosa, and Arabian. Colts were overrepresented compared with fillies, 89/146 and 57/146, respectively, which was true for each group of foals from both seasons (Table 1).

There was no significant difference in survival status for age, sex, or breed (Thoroughbred versus non-Thoroughbred) in the foals of season 1 or 2. Similarly, the survival rate in hospitalized foals treated at either referral institution was not different for both seasons (OR 1.4; 95% CI, 0.56–3.3 and OR 1.15; 95% CI, 0.22–4.9, respectively). The overall survival rate for all foals was 67% (98/146), where septic foals had the lowest survival rate (40/82, 49%) compared with sick nonseptic (34/40, 85%) and healthy foals (24/24, 100%) (Table 2).

PTH, PTHrP, and CT Concentrations

For each group of foals in season 1, PTH, PTHrP, and CT concentrations were determined, and for foals in season 2, only PTH concentrations were measured (Table 3). Septic foals had significantly higher PTH concentrations compared with sick nonseptic and healthy foals (P < .05). This was true in foals from both foaling seasons. There was no significant difference in PTHrP concentration between groups in season 1 foals. Plasma CT concentrations were significantly lower in hospitalized foals (septic and sick nonseptic foals) compared with healthy foals in season 1 (P < .05), however, for septic foals no difference was detected between survivors and nonsurvivors.

Table 3.   Blood hormone concentrations in neonatal foals from 2 foaling seasons (values expressed as median and range unless otherwise stipulated).
Foal ClassificationPTH
(pmol/L)
PTHrP
(pmol/L)
CT
(nmol/L)
  • **

    P < .05 comparing median values between each group of foals within an individual season.

  • PTH, parathyroid hormone; PTHrP, parathyroid-related peptide; CT, calcitonin; NA, not applicable. Total = average values for season 1 results and season 2 results.

Septic
 Season 1 (n = 47)7.4 (0–86)**0 (0–4.1)0 (0–8.2)
 Season 2 (n = 35)25 (0.1–200)**NANA
 Total (n = 82)16.2NANA
Sick nonseptic
 Season 1 (n = 24)5.5 (0–73)**0 (0–2.8)0 (0–2.7)
 Season 2 (n = 16)11 (0.5–48)**NANA
 Total (n = 40)8.25NANA
Healthy
 Season 1 (n = 12)1.1 (0–40)**0 (0–2.6)2.6 (0–20)**
 Season 2 (n = 12)5.3 (0–18)**NANA
 Total (n = 24)3.2NANA

TCa, Ca2+, Mg2+, and Phosphorus Concentrations

Because sample handling methods were different between the 2 foaling seasons for the determination of Ca2+ and Mg2+ determinations, each season is reported separately.

Foaling Season 1

There was a significantly higher TCa concentration in hospitalized foals (septic and sick nonseptic) compared with healthy foals (P<.05) (Table 4). Serum phosphorus concentration was significantly higher in septic foals compared all other foals (P<.01), and there was no difference in Mg2+ concentration among all groups of foals, although a number of septic foals (2/47) were hypomagnesemic (Mg2+<0.4 mmol/L). Serum Ca2+ concentration was correlated with serum Mg2+ concentration (r= 0.54, P<.01), serum PTH concentration (r=−0.48, P < .01), and total calcium concentration (r= 0.4, P < .01).

Table 4.   Serum total calcium (TCa), ionized calcium (Ca2+), ionized magnesium (Mg2+), and phosphorus concentrations in neonatal foals (values expressed as median and range unless otherwise specified).
Variable SeasonSepticSick NonsepticHealthyANOVA P Value
12121212
(n = 47)(n = 35)(n = 24)(n = 16)(n = 12)(n = 12)  
  • *

    Significance between groups for an individual season.

  • NP, not assessed. NA, not applicable.

TCa
(mg/dL)
1212121311NP.022*NA
(7.5–17)(7.6–16)(8.7–14)(10–15)(9.2–13)   
Ca2+
(mg/dL)
5.65.65.86.15.96.44.75.0086*
(3.1–7.6)(2.9–8.9)(3.5–7.4)(5.5–6.8)(4.1–7.0)(5.6–8.1)  
Phosphorus
(mg/dL)
6.9*7.25.35.36.3NP.0007*NA
(3.4–20)(3.8–22)(4.1–15)(3.2–12)(4.4–8.4)   
Mg2+
(mmol/L)
0.620.570.570.540.620.58.24.273
(0.39–0.95)(0.31–1.0)(0.38–0.81)(0.3–0.8)(0.48–0.79)(0.42–0.78)  

Foaling Season 2

Septic foals had a significantly lower Ca2+ concentration than all other foals (P<.01), with a median Ca2+ concentration of 5.6 mg/dL (range, 3.1–7.6 mg/dL) (Table 4). There was no significant difference in the Mg2+ concentrations between groups of foals (septic, 0.3–1.0 mmol/L; sick nonseptic, 0.3–0.81 mmol/L; healthy, 0.42–0.78 mmol/L; P= .27), but 5/35 septic foals had hypomagnesemia (Mg2+ < 0.4 mmol/L). In hospitalized foals, serum Ca2+ concentrations were correlated with TCa concentrations (r= 0.63, P < .01) and serum PTH concentration (r=−0.41, P < .01), but not Mg2+ (r= 0.12, P= .19).

Survival

Descriptive and Clinicopathologic. In both seasons, univariate analysis for survival was used to determine crude OR for survival for various descriptive and clinicopathologic variables (Tables 5 and 6).

Table 5.   Univariate analysis for survival among 84 neonatal foals (foaling season 1) including statistically significant specific clinicopathologic and descriptive data.
Variable (units)RangeCrude Odds
Ratio for
Survival
95%
Confidence
Interval
  • *

    P < .05.

Hematology
 Total leukocytes
(× 109/L)
0.3–4.00.360.09–1.26
4.1–12.02.7*1.39–9.3
12.1–35referent 
 Band neutrophils
(× 109/L)
0.05.3*1.35–21.3
0.01–0.30.70.22–2.34
0.31–2.2referent 
Biochemistry
 Parathyroid hormone
(pmol/L)
0.0–3.05.8*1.93–12.1
3.1–293.1*1.93–19.1
30–86referent 
 Creatinine
(mg/dL)
0.5–1.02.60.6–12.1
1.1–2.014.3*4.12–59.7
2.1–4.03.7*1.16–12.8
4.1–32referent 
 Fibrinogen
(mg/dL)
100–4003.5*1.23–9.9
401–1,140referent 
 l-Lactate
(mmol/L)
1.1–4.07.3*1.97–31.5
4.1–6.01.00.3–3.3
6.1–13referent 
 Glucose
(mg/dL)
81–1605.2*1.99–15.1
<80 or >161referent 
 Immunoglobulin G
(mg/dL)
16–4000.05*0.01–0.16
401–8001.060.13–22
>800referent 
 Potassium
(mEq/L)
2.4–4.53.64*1.43–9.7
4.6–7.9referent 
Descriptive data
 Mucous
membranes
Normal4.3*1.76–11.2
Abnormalreferent 
 Capillary refill
(seconds)
<24.7*1.67–13.8
24.4*1.16–19.5
3referent 
 Sepsis score0–≤1124.11*6.54–156.69
12 of higherreferent 
 Septic focus
present
No16.03*5.51–59.07
Yesreferent 
 Cold extremitiesNo12.2*4.5–37.3
Yesreferent 
Drug administration
 Pressor
administration
No9.7*1.2–200.3
Yesreferent 
Table 6.   Univariate analysis for survival among 63 neonatal foals (foaling season 2) including statistically significant specific clinicopathologic and descriptive data.
Variable (units)ValueOdds Ratio
for Survival
95%
Confidence
Interval
  • *

    P < .05.

Hematology
 PCV (%)15–354.95*1.1–28.7
36–453.170.9–11.7
46–58referent 
 Total leukocytes
(× 109/L)
0.3–4.01.50.25–9.7
4.1–12.05.5*1.03–32.8
12.1–35referent 
 Band neutrophils
(× 109/L)
0.05.0*1.3–20.1
0.1–0.31.140.26–5.0
0.31–2.0referent 
 Monocytes
(× 109/L)
0–0.250.18*0.05–0.6
0.26–0.6referent 
 Fibrinogen
(mg/dL)
100–40015.2*3.6–78.1
401–1,140referent 
Biochemistry
 Parathyroid hormone
(pmol/L)
0.0–3.02.620.67–11.1
3.1–2913.6*3.3–74
30–199referent 
 Phosphorus
(mg/dL)
3.4–6.05.33*1.4–24.1
6.1–8.02.70.6–12.9
8.1–20referent 
 Immunoglobulin G
(mg/dL)
16–4000.18*0.04–0.67
401–8000.15*0.03–0.7
801–2,756referent 
 l-Lactate
(mmol/L)
1.1–4.05.5*1.3–27.5
4.1–6.02.30.44–12.75
6.1–12.8referent 
 Anion gap6–136.0*1.33–32
13.1–203.140.71–15.8
21–43referent 
Descriptive data
 Temperature
(°C)
37.6–38.614.8*3.7–100.8
<37.5 or >38.7referent 
 Cold extremitiesNo12.5*3.5–60.3
Yesreferent 
 Pulse qualityNormal12*3.66–45.8
Abnormalreferent 
 Septic focusNo3.8*1.22–13.2
Yesreferent 

There was a strong association between sepsis score and survival status in these foals. In season 1, foals with a sepsis score of ≤11 were 24 times more likely to survive than foals with a score ≥12 (95% CI, 6.5–157). Septic foals in which a septic focus was not evident were 16 times more likely to survive than those with 1 or more foci of infection.

Foals that did not have cold extremities were more likely to survive than those that did (OR, 12.2; 95% CI, 4.5–37.3).

The presence of pink or pale pink mucous membranes with a capillary refill time of ≤2 seconds was associated with survival (Table 5).

In season 2 foals, a rectal temperature between 37.6 and 38.6 °C was associated with survival (OR, 14.8; 95% CI, 3.7–100.8) (Table 6). Nonsurviving septic foals had a significantly higher sepsis score than survivors (P= .02). Foals without an evident septic focus, without cold extremities, and normal peripheral pulse quality, were more likely to survive than those with a septic focus or foci, cold extremities or a weak or absent peripheral pulse (Table 6).

In season 1, foals with a serum creatinine concentration between 1.1 and 2.0 mg/dL were 14.3 times more likely to survive (95% CI, 4.1–59.7) compared with foals with a creatinine concentration >4.1 mg/dL (Table 5).

In season 2, monocyte counts of 0.0 to 0.25 × 109/L and the anion gaps of 6–13 were significantly associated with foal survival (OR, 0.18; 95% CI, 0.05–0.6 and OR, 6.0; 95% CI, 1.33–32, respectively), where as low monocyte counts and increased anion gaps were negatively associated with survival.

Total leukocyte count, band neutrophil count, fibrinogen concentration, IgG concentration, and l-lactate concentration were significantly associated with survival status in both seasons of foals (Tables 5 and 6).

Hormone Concentrations, Ca2+, Mg2+, and Phosphorus Concentrations and their Association with Survival. Serum PTH concentrations were significantly associated with foal survival in both seasons (Tables 5 and 6), where serum PTH concentrations were statistically higher in septic foals that died (P < .01) (median, range: surviving, 10.7 pmol/L, 0–86 pmol/L; nonsurviving, 41.1 pmol/L, 0–200 pmol/L). There was no statistical difference between blood culture-positive septic foals and blood culture-negative septic foals (P= .13). A PTH concentration between 0.0 and 3.0 pmol/L was significantly associated with survival in season 1 foals (OR, 5.8; 95% CI, 1.93–12.1) but not season 2 foals (OR, 2.62; 95% CI, 0.67 to 11.1). However, a PTH concentration between 3.1 and 29 pmol/L was associated with survival in both seasons (OR, 3.1; 95% CI, 1.93–19.1 and OR, 13.6; 95% CI, 3.3–74, respectively). For all 82 septic foals from both seasons, surviving foals (n = 40) had lower PTH concentrations than nonsurvivors (n = 42) when the data were combined (P < .01).

For season 1 septic foals, there was no association among PTHrP, CT, TCa, or Mg2+ (P > .15) concentrations and survival status. Serum phosphorus concentrations were significantly higher among nonsurvivors (P= .04).

For season 2 septic foals, there were no significant differences in TCa, Ca2+, or Mg2+ concentrations (P > .38) between foals that survived and those who died. There was a trend for higher serum phosphorus concentrations in nonsurviving septic foals (8.1 versus 6.8 mg/dL, P= .08).

Discussion

In this study, we found that serum PTH concentrations were associated with both foal sepsis and survival. Septic foals with increased PTH concentrations were more likely to die.

Increased PTH concentrations with hypocalcemia and normocalcemia are associated with death in critically ill humans.1,20,23 Similar findings were observed in the septic foals of this study where nonsurvivors had significantly higher PTH concentrations. Increased PTH concentrations have also been observed in adult horses with experimentally induced endotoxemia,3 naturally occurring enterocolitis,2 and rats with experimentally induced endotoxemia.24

Proposed mechanisms for increase in serum PTH release during sepsis include a physiologic adaptation to ionized hypocalcemia, inorganic hyperphosphatemia, or both,25 disturbances in Ca2+ sensing by the parathyroid gland, cytokine-mediated impairment of PTH-receptor responsiveness,3,20 catecholamine-mediated PTH hypersecretion from the parathyroid gland,1,26 and low blood pH.27

We found that PTH concentrations were increased in septic foals from both seasons; however, the interseason variability was high. We are unclear on the reason for this disparity, but we speculate that could be the result of different severity of illness among septic foals, which could translate into a variable inflammatory response in these foals. Sample handling was identical between seasons and, therefore, cannot explain this difference. Regardless, nonsurviving septic foals had increased PTH concentrations in both seasons, compared with surviving septic foals.

Increased PTH concentrations occur in critically ill human patients and in many instances appear to be independent of Ca2+ concentration.28 Previous studies have hypothesized that systemic inflammatory mediators and endotoxin stimulate the parathyroid gland to secrete PTH,3,24,29 which is speculated to occur in septic foals.

The cause for the increased PTH concentrations in septic foals reported here is likely multifactorial, where both mineral-related and nonmineral-related mechanisms are possible. Septic foals in this study had ionized hypocalcemia and hyperphosphatemia, both of which can stimulate PTH release. Some septic foals also had significantly increased creatinine concentrations suggesting hypovolemia, renal insufficiency, or both, which could contribute to these electrolyte derangements in the case of primary renal dysfunction; however, septic foals that died did not have more severe hypocalcemia or hyperphosphatemia than those who survived, suggesting that other nonmineral-related mechanism for increased PTH concentrations may be important.

Proinflammatory cytokines have been associated with parathyroid gland dysfunction in humans by altering PTH responsiveness on target cells.20,30 IL-8 has been identified as a specific modulating cytokine that is purported to induce PTH secretion,20,31 conversely, IL-1 and IL-6 have been shown to down regulate PTH secretion in the face of hypocalcemia in humans,20 cattle,32 and horses.33 Nonsurviving septic foals in this study might have had relatively higher concentrations of proinflammatory cytokines, eg IL-8 as previously reported,34 and measuring their blood concentration could have been useful to determine their association with PTH release, but such determinations were not the aims of the study.

Circulating catecholamines stimulate PTH secretion via a β-2 receptor agonist effect in humans.26 In exercising horses, where blood catecholamine concentrations are increased,35 studies have revealed that a physiologic hyperparathyroidemia, ionized hypocalcemia, and hyperphosphatemia also occurs.36–38 Blood catecholamines concentrations were not determined in the foals of this study; however, recent studies found that full-term septic foals have an intact hypothalamo-pituitary-adrenal axis that is greatly stimulated during early sepsis,8 indicating that stress responses are upregulated in the face of sepsis.

In our study, PTHrP was not significantly different among groups of foals and is unlikely to play a critical role in endocrine regulation of calcium homeostasis in septic foals.

Plasma CT concentrations were significantly lower in hospitalized foals, most notably septic foals. This is likely an appropriate response to ionized hypocalcemia and normocalcemia. Foals assigned a concentration of 0 nmol/L may actually have had circulating CT, however, if so, was below the limit of detection of the assay. Undetectable CT concentrations are not uncommon in normocalcemic animals, and despite the significant increase in CT precursors seen in the context of systemic inflammatory response syndrome and sepsis, mature CT concentrations do not increase.

Neonatal foals tend to have lower Ca2+ and TCa concentrations than adult horses15; however, the ionized hypocalcemia observed in these foals was associated with sepsis but not survival. While there was no difference in season 1, a difference was found in season 2 when a more stringent sample handling protocol was followed (ie, anaerobic conditions and analysis closer to the time of collection), confirming the importance of correct sample handling techniques to improve test accuracy.

Our values for Ca2+ and TCa in healthy foals were similar to another recent report.15

Ionized hypocalcemia has been observed in critically ill human patients with sepsis and negatively associated with survival.1,20,39,40 Numerous studies have revealed an association with critical illness, endotoxemia, and hypocalcemia in horses2,3,41–44 and other species.45 The prevalence of ionized hypocalcemia in critically ill horses is reported to be 75 to 86% of horses with enterocolitis,2 endotoxemia,3 or surgical colic.42

Proposed mechanisms for the development of hypocalcemia in sepsis include renal calciuresis,46 intracellular Ca2+ accumulation or extracellular ion shifts, tissue sequestration,2 chelation,28 impairment caused by poor target tissue responsiveness to PTH,47 or 1,25-D3, parathyroid gland dysregulation or dysfunction,2,3,20 failure to synthesize 1,25-D3, hypercortisolemia,48 and magnesium depletion.3,42,47,49 Studies investigating urinary excretion of electrolytes in septic human patients20 and endotoxemic horses3 suggest that hypercalciuresis does not occur in septic patients, making renal calcium wastage unlikely in septic foals.

Procalcitonin (PCT) is cleaved to CT where it is released from the thyroid C cells in health in response to hypercalcemia. PCT has no effect on calcium regulation, however, increase in blood PCT concentrations is inversely related to Ca2+ concentrations in septic human patients, and increase in PCT concentrations become more pronounced with the severity of disease.41,50–52 Recently, this has been questioned53 and unfortunately PCT concentration determinations are unavailable in horses.

Hypomagnesemia was observed in few septic foals but was not related to survival. Despite the administration of Mg2+-containing crystalloid solutions, some foals had Mg2+ concentrations <0.4 mmol/L, and unlike Ca2+, reduced dietary intake could be in part responsible for this finding.54 Hypomagnesemia is commonly reported among critically ill human patients21 and has been observed in adult horses administered endotoxin3 and hospitalized horses.4

Up to 48.7% of hospitalized horses have magnesium concentrations below the reference range, and horses with multiple organ failure or dysfunction were 2.31 times more likely to be hypomagnesemic and have longer hospital stays.4 Decreased Mg2+ is a cause of persistent hypocalcemia through impaired PTH secretion, end-organ PTH responsiveness, and decreased synthesis of 1,25-D355 and has been observed in horses and humans.

Healthy foals have higher Mg2+ and TMg concentrations compared with adult horses and our values for healthy foals were similar to those previously reported.15 Despite the presence of hypocalcemia in septic foals, concurrent hypomagnesemia was not observed in most foals. This could be attributable in part to a higher resting Mg2+ as is observed in neonates, insufficient time for the development of magnesium depletion, minor effect of administered Mg2+-containing crystalloid solutions or other unknown factors. In addition, higher PTH concentration would not be expected if magnesium concentrations were below normal in septic foals.

Several nonendocrinologic variables were significantly associated with survival in both populations of foals (Tables 5 and 6), and similar findings have been reported in other studies.5,6,8,11,12

In this study, we revealed that calcium dysregulation associated with increased PTH concentrations does occur in septic foals and that foals with higher PTH had a lower likelihood of surviving. We also documented that ionized hypocalcemia does occur in septic foals and is unlikely to be related to magnesium concentrations in early sepsis. We could not demonstrate any association with PTHrP with survival, yet hospitalized foals had lower CT concentrations.

Dynamic assessments of calcium regulatory hormones, Ca2+ and Mg2+ in septic foals over time may provide important species-specific information on calcium dysregulation in horses with prolonged disease. Additional studies are needed to determine the potential value or possible harm of calcium and magnesium supplementation in the treatment foal sepsis.

Footnotes

aCell-Dyn 3500R analyzer, Abbott Laboratories, Abbott Park, IL

bBoehringer Mannheim/Hitachi 911 system, Boehringer Mannheim Corp, Indianapolis, IN

cAccutrend Lactate analyzer, Roche, Mannheim, Germany

dACL 200 Automated coagulation laboratory, Instrumentation Laboratory, Lexington, MA

eNova 8+ Electrolyte analyzer, Nova Biomedical, Waltham, MA

fParathyroid hormone assay: I-PTH DSL-8000, Diagnostic Systems Laboratory Inc, Webster, TX

gParathyroid hormone related peptide assay: PTHrP DSL-8100. Diagnostic Systems Laboratory Inc

hCalcitonin assay: Ultrasensitive calcitonin DSL-5200, Diagnostic Systems Laboratory Inc

iPrism, version 4.0a GraphPad Software Inc, San Diego, CA

jSAS version 9.1, SAS Institute Inc, Cary, NC

kExcel, Microsoft Corporation, Mountain View, CA

Acknowledgments

We thank all of the technical staff and veterinarians at Hagyard Equine Medical Institute and The Ohio State University Galbreath Equine Center for their dedication and assistance for this project. Special thanks are extended to Dr Holly Aldinger for sample collection from healthy foals.

Funded by The Ohio State University Equine Research Funds and the Grayson Jockey Club Research Foundation.

Ancillary