Page 78 - JSOM Fall 2023

Blood samples were immediately placed on ice and centri- Figure 1C) and 219% (p < 0.001; Figure 1D; respectively)
fuged at 3,000 RPM (1,000 × g, 4°C) for 15 minutes; plasma higher than Pre values. On day 3, plasma adrenaline and nor-
was aliquoted and immediately frozen and stored at –80°C adrenaline concentrations at the Post 2 time point were 384%
for future analysis. All plasma samples were batch analyzed (p < 0.001; Figure 1C) and 68% (p < 0.001; Figure 1D; respec-
in duplicate for total testosterone (TT), cortisol, thyroid- tively) higher than Pre values. uc-Ocn was lower at the Post 1
stimulating hormone (TSH), triiodothyronine (T3), thyrox- and 2 time points compared to Pre by 30-38% (p < 0.001; Fig-
ine (T4), free triiodothyronine (fT3), free thyroxine (fT4) ure 1E) and 28–30% (p < 0.001), respectively, on both diving
(Calbiotech, CA, USA), as well as dehydroepiandrosterone days. Pre-dive c-Ocn concentration was 19% higher on day 3
(DHEA), insulin-like growth factor-I (IGF-1), adrenocortico- when compared to day 1 (p < 0.001).
tropic hormone (ACTH), insulin-like growth factor-binding
protein-3 ( IGFBP-3), sex hormone binding globulin (SHBG) FIGURE 1 Changes in stress-related hormones following
(ALPCO, Salem, NH), human thyroxine binding globulin cold-water dives in military personnel. Concentrations of
(TBG) ( MyBioSource, San Diego, CA), leptin (EMD Millipore, (A) Adrenocorticotropic Hormone (ACTH), (B) Cortisol,
(C) Adrenaline, (D) Noradrenaline, (E) Uncarboxylated Osteocalcin
St. Louis, MO), free fatty acids (FFA), (BioVision, Milpitas, (uc-Ocn), and (F) Carboxylated Osteocalcin (c-Ocn) were measured
CA), lactate (BioVision, Milpitas, CA), adrenaline/noradren- before (Pre) and after the first (Post 1) and second (Post 2) dive of the
aline (Diagnostika GMBH, Germany), bioactive osteocalcin day. The (G) molar ratio of circulating Adrenaline to Noradrenaline
(Ocn) (BioLegend, San Diego, CA), and carboxylated osteo- (Adrenaline/Noradrenaline) was calculated at each time point. Each
calcin (c-Ocn) (Takara Bio USA Inc., San Jose, CA) via en- circle represents one participant’s value (n=5), and the bars represent
the mean concentration for each time point.
zyme-linked immunosorbent assay (ELISA). For the c-Ocn
analysis, 500µL plasma of each sample was converted into
serum prior to analysis. 25
Statistical Analyses
Statistical analyses were performed using R 3.6.1 (R Core
26
Team) and the Rallfun-v37 package as described. To assess
the impact of cold-water diving on hormonal and physiologi-
cal measures, within-by-within analyses were performed using
20% trimmed means and Winsorized variances and covari-
ances for dependent groups with day (day 1 vs. day 3) and
time (pre vs. post 1 vs. post 2) as factors. Additionally, pair-
wise comparisons of the difference scores between Winsorized
means were performed using a percentile bootstrap method
for dependent groups and Hochberg’s method to control the
family-wise error rate. Statistical significance was accepted at
p < 0.05. All data are expressed as mean ± standard error of
the mean.

Results
Participants
Demographic information is in Table 1.

TABLE 1 Participant Demographics
Mean ± SE
*p < 0.05 vs. Pre; #p < 0.05 vs. Post 1, †p < 0.05 vs. respective value
Age (years) 32.4 ± 1.1 on day 1.
Height (cm) 180.9 ± 2.0
Weight (kg) 82.6 ± 2.9 Metabolic Hormones
Fat Free Mass (kg) 47.4 ± 4.1 A within-by-within analysis of metabolic hormone concentra-
Fat Mass (kg) 10.3 ± 1.6 tions uncovered a main effect of time (Pre vs. Post 1 vs. Post
VO max (ml/kg/min) 59.4 ± 1.7 2) for TSH and leptin; a main effect of day (day 1 vs. day 3)
2
BMR (kcals/day) 1955.9 ± 49.7 for fT3, T4, and leptin; and an interaction effect (day vs. time)
for T4 and fT4 (p < 0.05; Figure 2A–G). No effect of time
Stress-Related Hormones or day were uncovered for TBG concentrations (p > 0.05).
A within-by-within analysis of stress-related hormone concen- Pairwise comparisons also revealed that leptin was signifi-
trations uncovered a main effect of time (Pre vs. Post 1 vs. cantly decreased following both dives on day 1 and following
Post 2) for all stress-related hormones and an interaction ef- the first dive on day 3 (p < 0.001; Figure 2G) and the Post 1
fect (day vs. time) for uncarboxylated (un-Ocn) and c-Ocn and (p < 0.001) time points compared to day 1.
the adrenaline/noradrenaline ratio, but no differences between
days for any of the hormones (p < 0.05; Figure 1A–G). Pair- Growth-Related Hormones
wise comparisons revealed ACTH and cortisol concentration A within-by-within analysis uncovered a main effect of time
significantly decreased across both dives and days (p < 0.001, (Pre vs. Post 1 vs. Post 2) for testosterone (p < 0.001; Figure
Figure 1A–B). On day 1, plasma adrenaline and noradrenaline 3A), IGFBP-3 (p < 0.002; Figure 3D), and SHBG (p < 0.05;
concentrations at the Post 1 time point were 173% (p < 0.001; Figure 3E); a main effect of day (day 1 vs. day 3) for IGF-1

76 | JSOM Volume 22, Edition 3 / Fall 2023